Technical Support

Recombinant Proteins FAQ

• How recombinant proteins are produced?

  Recombinant proteins are proteins encoded by recombinant DNA cloned in a system that supports gene expression and mRNA translation. The host cells used for recombinant proteins production can be derived from bacteria, mammalian cell, insect and yeast.

• What is the function of ROX in SYBR Green qPCR Master Mix series kit?

  In the qPCR reaction, the difference of fluorescence signals will be caused by the slight difference of the volume of reaction solution, the internal difference of qPCR tubes, and the random factors during the reaction. Therefore, the same amount of Rox dye is added to each reaction well, and its functions include：
  1) Correct the signal change caused by foam or evaporation in a single reaction.
  2) Corrects the intra-batch volume difference caused by the difference of pipettes.
  3) Correct the volume difference between batches, providing more continuous and consistent repeated Ct values between batches.

• What is the product of reverse transcription?

  It is cDNA (complementary DNA), a DNA strand that is complementary to RNA strand in base pairing. Our RT mix can efficiently synthesize the first strand cDNA.

• What are the shipping conditions for recombinant proteins?

  Most recombinant proteins are shipped at blue ice or room temperature. Lyophilization improves product stability and reduces packing materials and shippingcosts. Data fromquality control tests indicates that lyophilized products shipped at ambient temperature retain full activity when delivered promptly and stored properly.

• What polymerase is used in mix?

  It is a hot-start Taq DNA polymerase, which is chemically modified. Its binding ligand can inhibit the activity of Taq DNA polymerase at room temperature. the ligand is completely dissociated and Taq DNA polymerase is fully activated, when the temperature exceeds 50℃, thus avoiding nonspecific amplification in the PCR, improving sensitivity and specificity of reaction, and enhancing stability of the product.

• What experiment is the cDNA used for?

  It can be used for qPCR and PCR. But it is not recommended for amplification of extra-long or extremely complex fragments.

• I ordered a vial of a pouch pack protein. Why it look like there is nothing in the vial?

  The volume of the lyophilized powder is affected by the composition before lyophilization, ionic strength, buffer type, product concentration, etc.The product appearance may become transparent, film-like and therefore unobservable. This is a common occurrence and we recommend using the product as usual. In addition, the lyophilized product has beendislodged from the bottom of the vial during shipment and dispersed on the vial wall or cap. A quick spin before opening the vial may bring the lyophilized product back to the bottom of the vial.

• Why is there no Ct value?

  1) There is an error in the procedure of detecting fluorescence signal: SYBR Green is generally collected at 72℃ extension.
  2) The degradation of primer or probe. Its integrity can be detected by electrophoresis.
  3) Insufficient amount of template. For samples with unknown concentration, Start with the highest concentration of serial diluted samples.
  4) Template degradation. Avoid the impurities and repeated freezing and thawing.

• What is the first element to obtain high quality cDNA?

  High quality total RNA. In general, the extracted RNA can be directly detected not the cDNA. The quality of extracted RNA decides the quality of cDNA produced by reverse transcription.

• How to choose a primary antibody?

  The selection of a primary antibody should meet the following three criteria:
  1. Target protein: Confirm the name of the target protein (verify Gene ID and UniProt ID).
  2. Reactivity: The antibody's reactivity should include the species of the sample to be tested.
  3. Application: Ensure the antibody's applications listed in the manual include the intended experimental use.

• How to determine protein purity and quantity?

  Methods for purity determination: a. SDS-PAGE; b. HPLC; c. Silver staining.
  Methods for protein concentration determination: a. Branford protein assay; b. BCA protein assay; c. Intensity measurement on the SDS-PAGE gel with a BSA standard curve.

• Why can't the Ct value be more than 35 or 38?

  1) Low amplification efficiency. Design a better primer or probe, and appropriately lower the annealing temperature.
  2) The reaction component is degraded or the sample amount is insufficient.
  3) The amplification product is too long. Generally, the product length is 80-250 bp.

• How to eliminate the influence of gDNA on PCR amplification?

  1) Avoid gDNA amplification in primer design.
  2) Total RNA was extracted and then treated with gDNA digester to remove gDNA.
  3) Select RT Master Mix for qPCR (gDNA digester plus) (Cat. No.: HY-K0511A) to eliminate the influence of gDNA on subsequent experiments.

• How do I reconstitute my recombinant proteins?

  Centrifuge the tube before opening
  1) During shipment, the protein may adhere to the wall or cap of the vial. Before opening the vial, please centrifuge at 10000-12000 rpm for 30 seconds to gather the protein at the bottom of the vial. If a high-speed centrifuge is not available, please centrifuge at 3000-3500 rpm for 5 mins.
  2）After centrifugation, add the reconstitution buffer to the lyophilized protein powder and mix gently by pipetting. Resuspend in the reconstitution buffer to recommended concentration (no less than 100 μg/mL).
  Note: Vigorous vortexing should be avoided as it can cause protein foaming and denaturation, thereby affecting the protein activity.
  3）Once reconstituted, recombinant proteins can be stored no more than a week at 2-8°C.
  For experiments with a short cycle (no more than 7 days), the recombinant protein solution can be directly added to the culture system and used up within a week. If the experimental concentration is lower than the reconstituted concentration, dilution can be done with a solution containing carrier proteins.
  4) For long-term storage, the protein solution should be diluted further with carrier proteins (0.1% BSA, 5% HAS, 10% FBS or 5% trehalose), and then aliquot and stored at -20°C to -80°C.
  It is not recommended to freeze the reconstituted product directly at -20°C to -80°C. Some recombinant proteins may stick to the plastic tube wall easily, which results in a lower concentration of protein in the solution and ultimately reduces its activity. Carrier proteins can prevent products from sticking to the tube wall by pre-blocking the protein binding site. Therefore, for long-term storage, cytokines or proteins should be further diluted with the solution containing carrier proteins before making aliquots and freezing.
  Note: Avoid repeated freeze/thaw cycles. Each freeze/thaw cycle will cause denaturation or conformational changes in some proteins, thereby reducing the binding ability of antibodies and accelerating protein degradation.

• Why does the melting curve have more than one main peak in qPCR?

  1) Primer design is not optimized. Avoid the appearance of primer dimer and hairpin structure, and avoid too much difference of annealing temperature between a pair of primers.
  2) Poor primer concentration. Reduce the concentration of primers appropriately and pay attention to the concentration ratio of upstream and downstream primers.
  3) The template is contaminated with genome. Avoid mixing gDNA during RNA extraction, and avoid nonspecific amplification through primer design.

• Why can't the products of RNA reverse transcription be observed by electrophoresis?

  The reverse transcription product is obtained by reverse transcription from the template. If the amount of template itself is low, the amount of reverse transcription will be even lower and the size is very uneven, so usually the reverse transcription products of RNA cannot be directly electrophoresed. For specific experiments, it is recommended that the experimental material and extracted RNA should not be stored for too long.

• We include a resolution buffer with the order; can I change the resolution buffer?

  No, the solubility of recombinant proteins is related to many factors, the most important of which are pH value and ionic strength. If the pH and ionic strength of the buffer you use do not suitable, resulting in insolubility of recombinant proteins.. MCE's cytokines or recombinant protein are strictly tested.

• What is the reason for the poor repeatability of the experiment in qPCR?

  1) Samples are not evenly mixed. 2) The error of sample volume is large. 3) There are bubbles when adding samples. 4) The temperature uniformity of the instrument is not good. 5) Template concentration is low. The lower the initial concentration of the sample, the worse the repeatability. Reduce the dilution ratio of the sample and set the concentration gradient to find the most reaction system.

• Can I use the vortex shaker to help the lyophilized powder dissolve fully?

  Proteins have a specific spatial structure that gives them optimal activity. During the dissolution process, It is recommended to use a pipette to gently blow and mix, or turn it upside down to mix.

• What is the specific activity of your recombinant proteins? What is meant by a “unit” of protein activity?

  The biological activity (ED ₅₀) (or “unit”) for each recombinant protein is available on the certificate of analysis. The ED ₅₀ is defined as the protein concentration at which the activity is 50% of the maximum response and is reported in ng/mL. This method of expressing activity should only be used for proteins whose dose-response curves are sigmoidal in shape. The formula for converting the activity as an ED₅₀ to specific activity is:
  
  
  Please note that MCE does not use the International Standard provided by WHO (National Institute for Biological Standards and control) for measuring recombinant protein activity. There is not a way to convert between these“International Units” and the ED₅₀. The best way to compare the activity of recombinant proteins from different sources is to do the same bioassay side-by-side using the same system.

• Do most proteins show cross-species activity?

  Species cross-reactivity must be investigated individually for each product. Many human cytokines will respond well in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have lower specific activity when used in the opposite species.

• Why are some proteins fused to tags? Do protein tags affect protein activity?

  Most MCE recombinant proteins are tag-free. Protein tagsare usefulin some siotuation.
  a. Protein tags are useful for protein purification.
  b. Tags are used for protein detection in Western blot or ELISA when the specific antibodies are not available.
  c. The Fc tag stabilizes molecules, which may increase the half-life of the linked products. Since the Fc fragment tends to dimerize, it helps the linked protein, particularly receptors, to form biologically active dimers.
  Protein tags generally do not affect the protein’s activity. For some small tags, such as the His-tag, do not need to be removed. There are more than 100 structures of His-tagged proteins in the Protein Data Bank,which indicates that the small His tags often do not usually interfere with correct protein folding. Additionally, tested activity results are listed on our protein web pages. If you have concerns about tags interfering with protein activity and there is no activity data online, please feel free to contact us for latest information at tech@medchemexpress.com.

Peptides FAQ

• What kind of documents will you provide for peptides?

  Usually we provide COA, HPLC and MS reports for peptides product.

• What is the difference between ‘peptide content’ and ‘peptide purity’?

  Impurities, salts and water will be brought in peptides during the product production process. Peptide content refers to the percentage of total peptides present in the product compared to everything else in the sample, such as water, salts, etc. Usually we determine peptide content through amino acid analysis or element analysis.
  While peptide purity, usually measured through HPLC analysis, is the percentage of peptides relative to peptide impurities, which does not take water and salts into account.

• How to determine peptide amount used in my experiment?

  Besides peptide, there are still salts, water and impurities present in final products. We can calculate theoretical amount (Net Weight) first, then use peptide content (%) and peptide purity (%) to calculate the actual amount (Gross Weight).
  1. Net weight = Concentration * Volume * MW (MW refers to free base molecular weight of peptide)
  2. Gross Weight = Net Weight ÷ (Purity% * Peptide Content%)
  

• What if I need the peptide that is not in your product list?

  MCE also offers unlimited Custom Peptide Service for our customers, you can submit your information in our website, or email/call us directly. We will send you feedbacks as soon as possible.

• How to restore peptides?

  Mostly we provide peptides in lyophilized powder form. If long-term storage is needed, peptides need to be stored at -20°C, dark and dry conditions in lyophilized powder form. Please store peptides with N2 or other inert gas if peptides contain amino acids like Met, Cys and Trp that are easy to oxidize. Also, peptides which contain easily degradable groups such as Asn and Gln are recommended to be used as soon. More, before using the peptide powder, please be sure that the test tubes are raised to room temperatures under dry conditions, preventing water from entering after opening the cap.

• How to dissolve peptides?

  Taking a small amount of peptides for solubility test is preferred since dissolution of peptides is kind of complicated. Also sterilized solvents are highly recommended for peptide dissolution.
  Normally peptide solubility depends on its sequence and modifications. You can calculate peptide length first. In general, peptides with amino acids fewer than six are soluble in water. For those containing more than 6 amino acids, the dissolution is mainly based on their total charge and hydrophobicity. You can refer to our peptide solubility guidelines.

• How to determine the stability of a peptide?

  The stability of a peptide is mainly determined by its sequence. Usually, peptides containing Cys, Met or Trp are easier to oxidize and need to be stored under oxygen-free conditions. Peptides containing Asn and Gln are more likely to degrade through deamidation. Still, peptides which contain Asp, Glu, Lys, Arg and His are easy to absorb moisture and deliquescence.
  Most peptides are easy to absorb moisture, so peptides are required to be stored under dry conditions. Repeated freeze-thaw cycles should be avoided.

Fluorescent Dye FAQ

• 1. There is no fluorescence appears under the fluorescence microscope after the incubation of the dye, what is the reason？

  1）Dye concentration is too low, you can appropriately increase the dye concentration
  2）incubation time is too short, according to the actual situation to adjust the incubation time
  3）Improper selection of fluorescence imaging channels to determine the specific excitation/emission wavelength of the dye
  4）Problems with the dye itself

• 2.What happened to the cells that died at the end of the dye incubation?

  1）Some of the final concentrations of dyes are too high and can cause toxicity to cells, resulting in cell death, so adjust the concentration of dyes according to the actual situation.
  2）improper dilution method of dyestuff, dyestuff needs to be configured first storage solution, the use of PBS and other solvents need to be diluted to the working solution concentration.
  3) Improper operation of the experimental process.

• 3.What is the difference between flow experimental staining and ordinary staining？

  There is little difference between flow staining and normal staining in terms of experimental operation, and both stains are used at the same concentration.

• 4.Can some dyes that can stain animal cells be used to stain bacteria?

  Most of the cell dyes can stain bacteria, the staining situation of different strains is different. Gram-negative bacteria have a cell wall thickness of about 10 nm, and the cell membrane structure is close, the overall permeability is good, the stain can easily penetrate into the cell membrane, showing a good cell membrane outline and a clear and complete structure. In contrast, Gram-positive bacteria showed unclear cell membrane outline and incomplete cell structure because the cell wall was about 20-80 nm thick and not close to the cell membrane structure, so the staining solution could not penetrate easily.

• 6.What need pay attention when staining for myofilament proteins？

  When fixing samples, fixation with fixatives containing methanol, which can damage the myofilament protein structure, should be avoided. Care should be taken when using it.

• 7.Some dye reagents have AM and no AM, what is the difference？

  The AM is an acetylmethoxy methyl ester (AM) group that is hydrophobic, allowing it to easily penetrate living cell membranes. Upon entering the cell, the AM on the dye is sheared by intracellular esterases to form a membrane-non-permeable polar molecule, which stays in the cell and retains fluorescence intensity。

• 8.What do the functional groups behind the labeled dyes mean？

  1）NHS/SE: collectively, succinimides are used to label free amines on antibodies, proteins, peptides, amine-modified oligonucleotides and other biomolecules (-NHX)Maleimide：
  2）Maleimide, used to label sulfhydryl groups on antibodies, proteins and peptides
  3）Tetrazine：Tetrazine-cyclooctene (TCO) for PROTOC
  4）Azide/alkyne azide - labeling of ethylene by click chemistry
  5）hydrazide - used to label aldehyde and ketone groups
  6）Carboxylic acid: for labeling amines after carbodiimide pre-activation, or for Steglich esterification of alcohols
  7) Amine - used to label various electrophile compounds, such as activated esters

• 9.Cells/tissues have been immunofluorescently labeled, can other labeling be done with dyes

  Yes, should be attention to avoid overlap between dye excitation/emission wavelengths and antibody fluorescence wavelengths as much as possible.

• 10.What are the special requirements for antibody/protein labeling using dyes

  1) The molecular weight of the antibody/protein is not less than 20 kDa
  2) Avoid using buffers containing primary amines (e.g. Tris, glycine) or ammonium ions, which compete with the protein to be labeled
  3) Low concentrations of sodium azide (≤3 mM or 0.02%) or thimerosal (≤0.02 mM or 0.01%) do not significantly interfere with protein labeling; however, 20-50% glycerol reduces labeling efficiency.

• 11. What is the ratio of protein/antibody label to dye usage?

  Usually the molar ratio is between 1:5 and 1:20 and the protein/antibody concentration is not less than 1 mg/mL.

• 12.How should the dyes be stored?

  It can be stored for one year from the date of opening at the storage temperature indicated on the product. For repeated use, it is recommended that DMSO be used to dissolve and dispense the product, which can be stored at -80°C for several months after dispense. Note that DMSO solutions are less stable and require anhydrous DMSO for dissolution and avoid freeze-thaw cycles.

• 13.Why is it necessary to use a solvent without Ca2+ or Mg2+ ions when dissolving the sodium or potassium salt of fluorescein?

  Mg2+ is an important factor in catalyzing the oxidation of fluorescence, Ca2+ is an ion related to the oxidation of luminalin, and the buffer is recommended to use DPBS to dissolve the best effect.

Isotope-Labeled Compounds FAQ

• What types of compounds are Isotope-Labeled compounds?

  One or more atoms are replaced by isotopic atoms in the molecular structure of compounds are called Isotope-Labeled compounds.

• What is the general application of Isotope-Labeled compounds?

  Isotope-Labeled compounds can be used as tracers or internal standards.

• Whether all the Isotope-Labeled compounds provided by MCE are stable isotope compounds?

  Yes, MCE currently offers Isotope-Labeled compounds are all stable Isotope-Labeled compounds.

• What are the stable Isotope-Labeled compounds that MCE can provide?

  MCE can provide stable Isotope-Labeled compounds containing deuterium (2H, D), carbon-13 (¹³C), nitrogen-15 (¹⁵N ), oxygen-17 & oxygen-18 (¹⁷O & ¹⁸O ), etc.

• Can MCE provide custom synthesis of Isotope-Labeled compounds?

  After evaluating, MCE can offer custom synthesis of Isotope-Labeled compounds contains different stable isotope atoms. For example, the type and quantity of isotopic atoms (such as D, ¹³C, ¹⁵N, etc) in the molecule should be evaluated to determine whether it can meet your scientific research needs.

• What kind of testing data of the Isotope-Labeled compounds will be provided by MCE to ensure the quality?

  MCE has a thorough quality control system and a robust quality management system. All the Isotope-Labeled compounds provide by MCE identified by qualified professional staff. All the compounds contain COA and analytical results ( generally include specific detection such as NMR, LC/GC-MS, HPLC & isotopic enrichment, etc ) to ensure the quality.

• Why does one compound has more than one corresponding Isotope-Labeled compounds?

  An labeled compound may contain multiple different isotopic atoms, even in different positions, so an unlabeled compound will have multiple different Isotope-Labeled compounds.

• How does MCE determine the Isotope-Labeled compounds’ purity and isotopic enrichment?

  Most of Isotope-Labeled compounds have been assigned a purity that is estimated based on NMR, LC/GC-MS, HPLC, etc. Each product is detected the isotopic enrichment through NMR or MS.

• Are MCE Isotope-Labeled compounds purities considered quantitative?

  Most Isotope-Labeled compounds are not considered quantitative. If some of our customers who want to know the potency of the products, we can provide additional test results such as TGA, Karl Fischer, etc. MCE does not list potency on products’ Certificate of Analysis (COA).

• Why does the appearance of the Isotope-Labeled compound received does not match the stated appearance on the COA?

  Some Isotope-Labeled compounds in salt form may be hygroscopic. Despite precautions taken, the hygroscopic nature of the compounds may change its appearance during transportation. Please note that this is only a change in physical properties and does not affect the chemical properties of the compound.

• What is the recommended storage temperature range?

  All of the Isotope-Labeled compounds’ storage conditions are indicated in the COA.

• How could we weigh and transfer some sticky oil compounds?

  General method: Small spatula, clean vial, analytical balance, gloves
  A) Clean vial is accurately weighed on analytical balance
  B) Dip the small spatula into the sample and place the sample into the clean vial, repeat until you get the desired amount.

Biochemical Assay Reagents FAQ

• Why are there multiple products with different names for the same CAS number?

  Some products in the form of hydrate and acid salt are still labeled with CAS number of free radical products. Therefore, it is recommended to choose products based on the structural formula.

• Why are there multiple CAS numbers for the same product?

  Because the products have different configurations, such as chiral, isome, etc.

• What is the difference between compounds with or without salt?

  The main structure of compounds with or without salt is the same, and the effect in biological experiments is the same, but the solubility will be different.

• Why I can't see the product in the bottle?

  Products sold in small sizes may not be easy to see because the product may adhere to the bottom or wall of the bottle. Centrifugation may be performed first and ensure that the solvent is in contact with all parts of the bottle.

• After receiving the product, the ice box in the package has melted. Will the product deteriorate?

  For some temperature-sensitive and unstable products, we will do additional packaging when the products are sent out to ensure product quality, and MCE guarantees the delivery time. If the ice box is melted when you receive the products, the quality of the products will not be affected in most cases. If the product has quality problems, you can timely contact the relevant technology or sales for processing.

• Is the product sterile?

  Most products are not aseptic packaging. If used in cell/animal experiments, please prepare well in advance. Most products can be dissolved in DMSO or provided in DMSO solution form. DMSO itself is strongly bactericidal and will not introduce bacteria to compounds. It is however important to keep the operating environment and the instrument be sterilized before experimental use. Compounds can also be sterilized by filtration prior to use depending on specific experimental requirements. High temperature and high pressure sterilization are NOT recommended.

• How to calculate when dissolving the product?

  Mass concentration (g/L) = molar concentration (mol/L) × molecular weight (g/mol)
  Molar concentration (mol/L) = mass fraction × density (g/L) / molecular weight (g/mol)
  Mass (g) = concentration (mol/L) × volume (L) × molecular weight (g/mol)
  Bulk liquid volume (mL) = concentration (mol/L) × preparation volume (L) × molecular weight (g/mol)/density (g/mL)
  Or you can use MCE Molarity Calculator and Dilution Calculator directly.

• What problems should be paid special attention to for some special products?

  For oily compounds, add DMSO to dissolve the compound and prepare the stock solution based on the volume without weighing. For unstable compounds, add nitrogen to the bottle when preparing the stock solution.

• How to prepare the stock solution?

  Select the appropriate solvent for the preparation of stock solution based on your experiment needs. Solubility information is available at the product webpage. Currently we only offer solubility data in DMSO and/or water, for solubility of other solvents, please email to tech@MedChemExpress.com. If you cannot find the solubility information you are looking for, or you are having difficulty preparing a product in solution form, you can also get help via the email above. Once prepared, aliquot the stock solution to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

• How do I dilute the compound solution for cell assay?

  Stock solution using ddH₂O as a solvent can be directly diluted with medium to prepare the working solution.
  When DMSO is used to prepare the stock solution, the stock solution is diluted in the culture medium to prepare a working solution. Make sure the concentration of DMSO is <0.5% to avoid poisoning the cells. A negative control in the experiment is usually the culture medium with DMSO at the same concentration. It is recommended that the process of dilution is performed in a stepwise manner to avoid compound precipitating caused by fast change of concentration.

Virtual Screening FAQ

• What is Virtual Screening (VS)?

  Virtual screening based on computer simulation technology, according to the three-dimensional structure or quantitative structure-activity relationship (QSAR) model of a specific target biomacromolecule, from the existing database of small molecule compounds, select the compound with strong binding ability to target biomacromolecules or in line with the QSAR model. Scientists can conduct biological experiments on selected compounds. Virtual screening is an initial step in drug development and an effective supplement to high throughput screening.

• What are the advantages of virtual screening?

  Virtual screening is based on the molecular docking computing technology of small molecular compounds and drug targets, which can quickly select active compounds with druggability from dozens to millions of small molecules. Virtual screening not only greatly reduced the number of experimental screening compounds (from 1 million compounds to 200 compounds), but also had short cycle, low cost and high positive rate.

• What are the types of virtual screening?

  There are two main types of virtual screening，one is structure-based virtual screening (SBVS) based on the structure of receptor biomacromolecules, the other is ligand-based virtual screening (LBVS). Receptor-based virtual screening is a virtual screening based on molecular docking. Its principle is to determine the binding of small molecules to receptors by means of molecular docking based on the three-dimensional structure of receptor biomacromolecules measured experimentally or homologously modeled, and evaluate the binding ability of proteins and small molecular compounds according to the affinity scoring function related to binding energy, and finally select compounds with reasonable binding modes and high predicted scores from a large number of compound molecules. The compounds obtained from these screens were finally used for subsequent biological activity tests. Ligand-based virtual screening is a virtual screening based on the pharmacophore model. Its principle is to establish a quantitative structure-activity relationship or pharmacophore group model based on the analysis of the structure, physicochemical properties, and activity relationship of existing drugs to predict and screen the activity of candidate compounds.

• What are the key elements of virtual screening?

  The key factors of virtual screening are to accurately determine or predict the molecular structure of the target protein, and to accurately and rapidly calculate the free energy change of the interaction between the candidate compound and the target. But these key factors are also bottlenecks that limit the accuracy of virtual screening.

• What does the MCE service include?

  MCE's virtual screening technology services include target research, molecular compound library preparation, molecular docking screening/pharmacophore modeling and screening, manual screening, and purchase/procurement of ideal physical compounds, etc.

• Positive rate of virtual screening?

  According to incomplete statistics, the positive rate of virtual screening is 5%-30%. Virtual screening has become the most potential tool for drug development, with more and more successful cases in drug design.

• What kind of compound library to choose for virtual screening?

  MCE's compound library is currently divided into known biologically active compounds and lead compound libraries, with 1.7w compounds in the known active compound library and more 1000w compounds in the lead compound library. In general, it is recommended to conduct virtual screening for the two categories of compounds together, so that the positive rate of matching will increase a lot. The first choice of compound library is as follows：1. It is recommended to screen the known active compound library (MCE Bioactive Compound Library (containing 15.6K compounds)), the compounds in this library (including Natural Product Library, FDA-Approved Drug Library, Drug Repurposing Compound Library, Traditional Chinese Medicine Active Compound Library, etc.) have superior performance in terms of IC50 and pharmacokinetics, so this library is the preferred compound library for virtual screening; 2. It is recommended to screen lead compound libraries with relatively novel structures in recent years (Discovery Diversity Set 50 (Containing 50.2K compounds)), the compounds in this library structure are relatively novel and highly innovative to the subject, therefore, this compound is selected for innovation-based subjects; 3. It is recommended to screen the diversity compound library (Life Chemicals 50K Diversity Library (containing 50.2K compounds)), the compounds in this library have various structures and are suitable for high-throughput screening.

• Are the screened compounds available in the market?

  The virtual screening compound library displayed on the MCE website, except for another national compound library (ID HY-L0093V), other compounds are physical compound libraries. Compound purchase is divided into MCE brand compound purchase and lead compound library purchase. The MCE brand compound purchase delivery period is 1 week, and the lead compound library purchase delivery period is 4-5 weeks. In general, the compounds in the physical compound library can be purchased, and individual hot-selling products or stranded compounds may be out of stock, and such compounds can be customized.

• What are the project report results provided by virtual screening?

  In general, the structural formula and physical information of the top 200 compounds in each compound library screened (three files include SDF, PDF, and Excel) are provided, and the docking pattern diagram of the top 5 compounds and the target protein is provided (2D/3D diagram), and PDF project report file (the report includes target protein background investigation, screening process, analysis of docking mode of 5 optimal compounds, etc.). If you have other reasonable requirements, please contact us in advance.

• Screening process/cycle.

  The virtual screening process includes preliminary research, preparation of compound database, preparation of protein structure, molecular docking calculation/pharmacophore modeling, etc. After receiving the order, the MCE team will immediately conduct research and start the project, combining software selection and manual selection The compound method takes a little time, but it can guarantee that the results obtained by analog screening are relatively good. The specific screening time is determined by the difficulty of the project, some are faster and some are slower, all in order to provide customers with an optimal result.

Inhibitory Antibodies FAQ

• What are monoclonal antibodies? What about their application?

  Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B lymphocytes against a particular antigen. They have many applications:
  1) Diagnostic tests: western blot, immunohistochemistry, immunofluorescence, etc.
  2) Analytic and chemical uses: ImmunoPrecipitation, CHIP, etc.
  3) Therapeutic uses: cancer treatment, autoimmune diseases, alzheimer's disease, COVID-19, etc.

• What are Inhibitory Antibodies?

  MCE Inhibitory Antibodies are research-grade biosimilar therapeutic monoclonal antibodies that have the same active biological component as the original therapeutic antibodies. Biosimilars make it possible to study the biological effects of a drug. These antibodies bind specifically to target cells or proteins, either stimulating the immune system to attack the malignant tumor cells or preventing tumor growth by blocking specific cell receptors. Target proteins include PD-1, PD-L1, EGFR, VEGFR, TNF-alpha, etc. MCE Inhibitory Antibodies have been extensively used in the most popular research fields, such as cancer, immunology and infection.

• What is the antibody structure?

  An antibody is composed of two heavy chains (50 KD each) and two light chains (25 KD each), which are joined by disulfide bonds to form a ‘Y’ shaped structure (150 KD). Antibodies are further divided into two regions: a variable region (Fab) and a constant region (Fc). 1) There are 5 types of heavy chain constant regions in antibodies (immunoglobulin) and classified into α-IgA, δ-IgD, ε-IgE, γ-IgG, μ-IgM. 2) There are two light chain isotypes: kappa and lambda.

• What the process of antibody humanization?

  1) Chimeric antibodies are structural chimeras made by fusing variable regions from one species like a mouse, with the constant regions from another species such as a human being.
  2) Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.
  3) Human antibodies are antibodies with fully human sequence. Usually MCE Inhibitory Antibodies can be used in human cell line, immunodeficiency mice, nude mice, humanized animal models or experimental conditions validated by the literature according to the species cross-reactivity.

• What is the Isotype Control? How do we choose the Isotype Control?

  An isotype control is an antibody used as a negative control to determine the amount non-specific background staining that can be observed. They are usually used as a negative control which making it easy for the researchers to differentiate between non-specific background effects or signal and specific effects of the antibody of interest. An isotype control should match the Inhibitory Antibodies as closely as possible. Select an isotype control that has the same host species, isotype, and label.

• What are the expression systems of Inhibitory Antibodies?

  Usually CHO stable cell line.

• How to determine the purity and quantity of antibodies?

  Methods for purity determination: a. SDS-PAGE; b. SEC-HPLC. Methods for protein concentration determination: a. Branford protein assay; b. BCA protein assay; c. Intensity measurement on the SDS-PAGE gel with a BSA standard curve.

• What are the shipping and storage conditions for Inhibitory Antibodies?

  Most Inhibitory Antibodies are shipped at blue ice or dry ice. Store strictly according to the storage conditions claimed on the certificate of analysis (COA) or product label. Keep in mind that every freeze/thaw cycle may cause some denaturation of the antibody.

• What is the appearance of Inhibitory Antibodies? Are they sterile?

  Usually, the inhibitory antibodies are supplied with format of liquid (exact concentration were shown on the product label and COA) or lyophilized powder. YES. They are sterile.

• How should I handle the Inhibitory Antibodies before use?

  During transportation, the inhibitory antibodies may adhere to the neck or cap of the vial. Before opening the vial, please centrifuge to gather the compound at the bottom of the vial.

• How can I prepare the stock solution or dilute it?

  If the appearance is liquid, they are already stock solution. Directly dilute the stock solution with medium, ddH2O or other buffer to prepare the working solution. If the appearance is lyophilized powder, in general, we recommend using ddH2O for reconstitution. Add the appropriate volume of ddH2O to the vial, and gently shake it to solubilize the antibody completely. Do not vortex. Then dilute the stock solution with medium, ddH2O or other buffer to prepare the working solution.

• Why the same products have two catalog No.? Such as HY-P9903 (Nivolumab) and HY-P9903A (Nivolumab (anti-PD-1)).

  Because the different catalog no. have different formulation, different storage and shipping condition.

Natural Products FAQ

• What types of compounds are natural products?

  If a product can find specific source information in the literature, it should be classified as a natural product.

• What are the commonly used detection instruments for natural products?

  Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) and high performance liquid chromatography (HPLC) or gaschromatography-mass spectrometry (GC).

• Are all natural products provided by MCE sourced from natural sources?

  No, some products are synthesized.

• Does MCE offer customized natural products?

  Yes, MCE will evaluate the customized needs of customers and respond accordingly.

Antibody-Drug Conjugates (ADCs) FAQ

• What are the data reports provided by ADC (Antibody-Drug Conjugate)?

  The CoA of ADC products includes SEC-HPLC, HIC-DAR, Endotoxin, Free-drug and other detection data.

• How to dissolve ADC?

  ADC solid powder contains excipients. It is recommended to dissolve all the ingredients when you get them and divide them into small bottles for storage to avoid repeated freezing and thawing.

• What is the functions of excipients in ADC?

  The functions of excipients in ADC include enhancing stability, improving bioavailability and promoting drug delivery, and play a vital role in maintaining the integrity of the drug during storage and use.

• Do the excipients in ADC affect the purity?

  Excipients do not affect purity or efficacy.

• What informations does the customer need to provide for a customized ADC?

  The customer needs to confirm the three main components of the synthesized ADC: antibody, linker, and payload. In addition, the customer need confirm DAR and preparation quantity.

Oligonucleotides FAQ

• What method is used to synthesize oligonucleotides?

  The phosphoramidite method is the method of choice for oligonucleotide synthesis. Phosphoramidite oligo synthesis proceeds in the 3'- to 5'-direction (opposite to the 5'- to 3'-direction of DNA biosynthesis in DNA replication).

• How to determine the oligonucleotide content?

  The oligonucleotide content is typically measured using ultraviolet-visible spectrophotometry (UV-Vis). Besides oligonucleotides, there are still salts, water and impurities present in final products. It is recommended to fully dissolve all powder, and then repackage it into smaller portions.

• What are the shipping conditions for oligonucleotides?

  The oligonucleotides are shipped at blue ice or room temperature. MCE provides you with oligonucleotides in lyophilized powder form. Lyophilized products shipped at ambient temperature retain full activity when delivered promptly and stored properly.

• What are the storage conditions for oligonucleotides?

  Upon receipt, store at -20°C. Make sure to always tighten the cap on the tube prior to storing. Lyophilized powder is easy to absorb moisture. Stored under these conditions, the product remains stable for one year.

• What if I need the oligonucleotide that is not in your product list?

  MCE offers unlimited oligonucleotide synthesis service, you can submit your information (sequence, purity, chemical modification and site) in our website, or email/call us directly. We will send you feedbacks as soon as possible.

Reference Standards FAQ

• Can you supply standards for LC/MS analysis?

  Yes, MCE offers a wide variety of reference standards (analytical standards), each rigorously tested under the ISO 17025 system. Comprehensive analytical documentation accompanies each product, providing evidence of purity, content, and structure, which supports measurement traceability.
  We use various detection methods (HPLC, GC, MS, NMR, etc.) for structural identification and purity assessment. Additionally, we employ TGA (Thermogravimetric Analysis), LOD (Loss on Drying), KF (Karl Fischer), and ROI (Residue on Ignition) for measuring loss on drying, residue after dissolution, moisture content, and ash content, respectively. Content calculation is performed using a mass balance method.
  

• Are the standards you supply certified reference materials (CRM)?

  No, we do not supply CRMs; we only provide reference standards that are rigorously tested under the ISO 17025 system.

• Can you supply standards customization service?

  Yes, we can supply standards customization service. All testing services are under the ISO 17025 system.

Compound Screening Libraries FAQ

• What information can be provided by the compound library ?

  A. Paper Compound Layout Information：Physical layout details included in printed form.
  B. USB Flash Drive: Contains three types of files:
  1. Excel File: Arrangement information of the compound library; Physical and chemical properties of compounds; Biological information related to the compounds.
  2. PDF File: Visual representation of the layout information for the library.
  3. SDF File (Structure Data File): Structural information for each compound available in the library.
  

• How to store the compound library and how long is the storage life?

  Storage Recommendations Temperature:
  1. -80°C: Ideal for long-term storage of compounds. Can be stored for up to 2 years from the date of receipt.
  2. -20°C: Suitable for shorter-term storage, with a maximum shelf life of 1 year from the date of receipt.
  If you buy a large volume compound library, you can aliquote the library after receiving the library to reduce the effect of repeated freezing and thawing on the compound.
  

• Do you offer customized compound libraries ?

  MCE offers highly customized libraries to suit your specific research requirements:
  1. Compound Types: Choose from a diverse range of products (eg: Approved drugs, novel compounds, natural products, metabolites, etc).
  2. Supply Forms: Powder or Solution.
  3. Plate Map: Flexible arrangements to meet your experimental needs.
  4. Quantities: Specify the exact amount you require (30/50/100/200 μL or 1mg).
  5. Packaging Choices: Plates (96/384-well plate) or 96-well Tubes
  You can also provide your specific requirements, there will be professionals to match the corresponding compounds for you.
  

• What are the specifications of the compound library ?

  Products from the compound library are available in both solution and powder form. Among them, solution products are mainly provided in 10 mM, compounds with product solubility of less than 10 mM are provided in 2 mM, and some compounds with uncertain molecular weight (eg: polymers, mixtures, etc.) are provided in 3 mg/mL concentration.
  Most solution products use DMSO as the solvent. A small number of compounds with poor solubility in DMSO will be dissolved in water or ethanol according to the actual solubility.
  

• What packaging format is the compound library ?

  For solution compounds: 96/384-well plates or 96-well tubes.
  For powder compouds: 96-well tubes.
  

Enzymes FAQ

• Can the units of enzymes from different companies be converted?

  For the same enzyme from different companies or different catalog numbers, unit conversion is only possible if the enzyme activity assay methods are identical. If the assay methods are different, direct conversion cannot be performed.

• If the enzyme is labeled in enzyme active units,how to calculate the protein/solid mass?

  The 100U in the product specification represents the total activity of the product. The specific unit definition and enzyme activity data are generally provided in the product's Certificate of Analysis (CoA). The test results can be viewed by downloading the corresponding test report from the official website according to the batch number of the purchased product.
  Calculation of product weights in two situations:
  1) When enzyme activity unit is expressed as U/mg protein
  For example, regarding the products in this batch of HY - P2804 #277748, the enzyme activity detection result indicates that the enzyme activity per milligram of protein is 104 U/mg protein. In other words, each milligram of protein has a vitality of 104 units. To calculate the protein mass corresponding to 100U, we perform the calculation 100÷104≈0.96 mg. Meanwhile, the protein content tested in this batch is 96%, meaning that protein mass accounts for 96% of the total solid mass. Thus, to determine the total solid mass of the tube of products you received, we calculate 0.96÷0.96≈1 mg.
  2) When enzyme activity unit is expressed as U/mg soid
  For example, HY-P2802 #293739 The enzyme activity detection result of this batch of products is 50.5 U/mg solid, that means the activity per mg of solid is 50.5 units, and the corresponding solid mass of 100U is 100÷50.5≈1.98 mg.
  

• How to dissolve enzyme?

  1) The website features products with “Solubility Data.” For reference, these data are based on our actual measurements and serve as a testament to the product quality;
  2) For some products, relevant solubility information can be found on the website page or in the Data sheet. This information is sourced from literature and can serve as a reference;
  3) If the information cannot be found through the above methods, it is advisable that a small amount of the enzyme be first attempted to be dissolved in distilled water. Alternatively, a dissolution plan could be developed based on your experimental requirements, with reference to similar cases and through the conduct of preliminary exploratory experiments. This will facilitate the acquisition of solubility information.
  

• How should the dissolved enzyme be stored?

  It is recommended to store the dissolved enzyme at 2-8°C for short-term use to avoid the impact of freeze-thaw on enzyme activity. For long-term storage, it is recommended to store at -20℃ according to the single experiment quantity.

• In case precipitation or flocculent substances appear after the enzyme has been dissolved

  1)Improper solvent selection: In some cases, improper solvent selection may lead to precipitation or flocculent after enzyme dissolution. For example, the addition of organic solvents may reduce the dielectric constant of the solvent, thereby increase the electrostatic attraction between solute molecules, and causing aggregation of enzyme molecules.
  2)Improper dissolved pH/temperature: As a protein with catalytic action, the stability of enzymes is affected by pH and temperature. When it deviates from the optimal range, the enzyme may undergo denaturation.This leads to a reduction in its solubility, consequently resulting in the formation of precipitation or flocculent substances.
  3)Excessively high salt concentration: An excessively high salt concentration in the solution can reduce the protein solubility, causing the protein to precipitate out of the solution.
  4)Low purity: Some naturally extracted enzyme products are provided in a crude form. These products may contain impurities that have not been completely removed, which may lead to the presence of insoluble substances upon dissolution.
  5) Insoluble protective agents are added: Some enzymes are susceptible to inactivate. To maintain stable activity, insoluble protective agents are added during the production process.
  6) Immobilized enzymes: Immobilized enzymes are insoluble in water and can be directly added to the reaction system for use.
  If there is precipitation/flocculent after dissolution,
  1) The solvent, pH, and temperature selected could be considered first;
  2) If the dissolution conditions are appropriate, please try diluting with distilled water;
  3) If no change is observed upon dilution with distilled water, it is recommended to use the supernatant for the experiment. If removal of impurities is necessary, filtration using filter paper is suggested.
  

Antibodies FAQ

• What do the abbreviations in the "Application" section of antibodies represent?

  IHC-P: Immunohistochemistry-Paraffin
  IHC-F: Immunohistochemistry-Frozen
  ICC/IF: Immunocytochemistry/Immunofluorescence
  mIHC: Multiplex Immunohistochemical
  IP: Immunoprecipitation
  ChIP: Chromatin Immunoprecipitation
  FC: Flow Cytometry
  ELISA: Enzyme Linked Immunosorbent Assay

• How should antibodies be stored?

  Most of antibodies should be stored at -20°C. The operation of Centrifugation and aliquoting are recommended upon receipt to avoid repeated freeze-thaw cycles. Some antibodies require storage at 4°C, please refer to the product manual for specific condition.

• How to choose a secondary antibody?

  1. Species origin: Select based on the species of the primary antibody. For example, if the primary antibody is from mouse, choose an anti-mouse secondary antibody (host species can be goat, rabbit, etc.).
  2. Label selection: Choose the secondary antibody's conjugated label based on the experimental type.
  Commonly used tag types are as follows:
  a. WB/ELISA: Enzyme (HRP/AP), Biotin, Fluorescent
  b. IHC: Enzyme (HRP/AP), Biotin
  c. IF/ICC: Biotin, Fluorescent
  d. FC: Biotin, Fluorescent
  

• What does "YA***" after the antibody name represent?

  "YA***" after the antibody name represents the clone number of the antibody. The clone number is used to distinguish different monoclonal antibodies. Only antibodies with the same clone number have identical antigen epitopes.

• Why do different antibodies have varying concentrations? What if the concentration does not meet experimental needs?

  Different antibodies have different affinities. MCE antibodies are formulated based on reactivity rather than concentration to ensure optimal experimental results. We guarantee that the antibodies will work at the recommended dilutions for the applications and species listed on the website.

• How to choose directly labeled and unlabeled antibodies?

  Directly labeled antibodies are conjugated with labels (e.g., HRP, FITC) on the primary antibody, avoiding non-specific binding from secondary antibodies and offering higher specificity. They also eliminate the need for secondary antibody incubation, saving time and simplifying the process.
  Unlabeled antibodies require secondary antibodies for detection. Since multiple labeled secondary antibodies can bind to a single primary antibody, they offer higher sensitivity, making them more suitable for detecting low-abundance proteins. Additionally, unlabeled antibodies are cost-effective and flexible for multiple labeling combinations.
  In a word, the choice between directly labeled and unlabeled antibodies could be based on experimental requirements and cost considerations.

• How to determine whether an antibody is monoclonal or polyclonal? How to choose?

  The "Clone Type" on the product page indicates the antibody type. Selection should be based on experimental needs.
  Monoclonal: Monoclonal antibodies are highly specific, targeting a single epitope on the antigen.
  Polyclonal: Polyclonal antibodies offer higher sensitivity as they recognize multiple epitopes on the antigen, making them more tolerant to conformational changes.
  Recombinant: Recombinant antibodies are produced through controlled processes, ensuring high batch consistency and avoiding issues like gene loss or mutation in hybridoma cells.

• Can I use an antibody if the application or species I need is not listed in the manual?

  Applications and species not listed are either not applicable or untested. It is generally not recommended to use the antibody for these purposes. Please contact technical support via email for additional information.

• Why does the molecular weight in WB not match the expected value?

  1. Post-translational modification: Phosphorylation, glycosylation, ubiquitination, etc.
  Band Change : larger
  Solution:
  ①Check databases or literature for modifications.
  ②Treat samples with appropriate reagents (e.g., PNGase F for deglycosylation).
  2. Formation of multimers: Bands appear at multiples of the monomer size.
  Band Change : larger
  Solution:
  ①Extend boiling time.
  ②For multi-pass membrane proteins, try gentle boiling or no boiling.
  3. Relative charge: Affected by amino acid composition.
  Band Change : larger or smaller
  Solution:
  Check databases or literature for effects on band migration.
  4. Isoforms or splice variants Target protein may have multiple isoforms or splice variants.
  Band Change : larger or smaller
  Solution:
  Review literature and check immunogen sequence to determine if the antibody recognizes these variants.
  5. Inappropriate gel concentration : Gel concentration affects band migration.
  Band Change : larger or smaller
  Solution:
  Adjust gel concentration: use lower concentration for larger proteins and higher concentration for smaller proteins.
  6.Protein degradation: Degradation results in lower molecular weight or multiple bands.
  Band Change : smaller
  Solution:
  Use fresh samples and add protease inhibitors.
  

• What to do if no target band appears in WB?

  1. Insufficient loading: Low protein concentration .
  Internal Control Band: None
  Solution: Measure protein concentration using BCA/Bradford assay; load 20-50 µg per lane.
  2. Incomplete transfer: PVDF membrane is hydrophobic and requires methanol activation.
  Internal Control Band: None
  Solution: Activate PVDF membrane with methanol before transfer.
  3. Incorrect membrane cutting: Some proteins have modifications or multimers.
  Internal Control Band: Present
  Solution:
  ①Research protein modifications in databases.
  ②Refer to literature for expected band positions and adjust cutting range.
  4. Low protein expression: Target protein is expressed at low levels in cells or tissues.
  Internal Control Band: Present
  Solution:
  ①Refer to literature for methods to increase target protein expression.
  ②Use qPCR to check mRNA expression levels.
  

• Why do multiple bands appear in WB?

  1. Target protein characteristics: Target protein has isoforms, multiple modifications, dimers, or multimers.
  Solution:
  ① Research target protein information in databases or literature.
  ② Boil samples for 10 minutes before loading if multimers are present.
  2. Sample issues: Cell lines with excessive passages may have altered protein expression profiles; protein degradation.
  Solution:
  ①Use low-passage cell lines as controls.
  ②Use fresh samples and add sufficient protease inhibitors.
  3. Blocking: Insufficient blocking.
  Solution : Re-prepare blocking solution (e.g., 5% skim milk/BSA) and extend blocking time.
  4. Antibody incubation: High antibody concentration or non-specific binding.
  Solution :
  ① Reduce antibody concentration or incubation time.
  ② Replace antibody or use 5% skim milk/BSA as dilution buffer.
  

PROTAC FAQ

• 1. Can you supply PROTAC synthesis services?

  Yes, MedChemExpress (MCE) can provide one-stop services for the design, synthesis, analysis, purification, optimization, detection, and evaluation of PROTAC-related products (Ligand for E3 Ligase, PROTAC Linker, Ligand for Target Protein for PROTAC, E3 Ligase Ligand-Linker Conjugate, Target Protein Ligand-Linker Conjugate, PROTAC, SNIPER, and PROTAC-Linker Conjugate for PAC).

• 2. What information do we need to supply before the PROTAC design?

  Please provide the target protein, the ligands for the protein if available, and the requirements for the linker and E3 ubiquitin ligase.

• 3. What degradation rate can the PROTAC molecules you design and synthesize achieve?

  We do not guarantee the activity but can provide the quality control report for the product itself, including NMR, HPLC, MS, etc.

• 4. What’s the cost of the PROTAC?

  The price of the PROTAC depends on the structure and quantity of the PROTAC.