3X FLAG peptide TFA 

3X FLAG peptide TFA is a FLAG-tagged peptide containing three repeats of the Asp-Tyr-Lys-Xaa-Xaa-Asp motif. 3X FLAG peptide TFA can be used for protein separation and purification, and competitive elution with target proteins.

1. Purification of FLAG tagged proteins through 3X FLAG peptide competition^[2]
(1) Plasmid transfection: Transfect plasmids of mIRS-1, hIRS-1, or their mutants labeled with FLAG into 293 T cells for 2 days.
(2) Cell collection: Cells were harvested by centrifugation at 14000 rpm for 10 minutes at 4 °C, and lysed with HEPES buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.2% sodium fluoride, 0.1% sodium orthovanadate, and protease inhibitor mixture).
(3) Using Anti-FLAG magnetic beads for protein separation and purification: Add Anti-Flag magnetic beads (HY-K0207)/Anti-c-Myc magnetic beads (1 μm) (HY-K0207A) to the cell lysate, and incubate at room temperature for 2 hours or at 4 °C overnight. Then wash with a washing buffer and elute with a buffer containing 1 mg/mL 3X FLAG. Finally, use a gel filtration column for further purification of the supernatant, which is equilibrated with the storage buffer. All purified proteins are concentrated using centrifugal filtration and aliquoted for storage at -80 °C.
(4) Protein quantification and validation: The purified protein was quantified using an ND-2000 NanoDrop spectrophotometer with OD 280 and validated by Coomassie Brilliant Blue staining.
2. Co-IP and co-IP-MS analysis using the 3X FLAG peptide^[3]
(1) Centrifuge the cell lysate at 13300 rpm for 15 minutes at 4 °C to remove intact cells.
(2) The supernatant was incubated overnight with control immunoglobulin G (IgG) or primary antibody in immunoprecipitation buffer (150 mM NaCl, 20 mM HEPES, pH 7.4, 1% Triton X-100, 12.5 mM β - glycerophosphate, 1.5 mM MgCl₂, 2 mM EGTA, containing phosphatase and protease inhibitors), and then incubated with 20 μL resuspended A/G protein beads at 4 °C for 2 hours to precipitate bound proteins.
(3) For continuous immunoprecipitation, incubate 3X FLAG eluent solution (final concentration 150 ng/mL) with 5 times the volume of gel at 4 °C for 2 hours, elute the bound protein from beads, and then use secondary antibody immunoprecipitation at 4 °C overnight. The beads were centrifuged at 1000 g for 5 minutes at 4 °C to remove the supernatant, washed 4 times with immunoprecipitation buffer, and boiled at 95 °C for 20 minutes.
(4) The samples were run on SDS-PAGE gel and then dyed (Bio Rad). Subsequently, cut off the tape, decolorize, digest with trypsin, and perform MS analysis to determine individual proteins using liquid chromatography-MS.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

2975.84

C₁₂₂H₁₇₀F₃N₃₁O₅₁S

Solid

White to off-white

Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys

MDYKDHDGDYKDHDIDYKDDDDK

Room temperature in continental US; may vary elsewhere.

Sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

• [1]. Zilio N, et al. Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe. Cold Spring Harb Protoc. 2017 Mar 1;2017(3):pdb.prot091611.  [Content Brief]

  [2]. Gao XK, et al. Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes. Cell Discov. 2022 Jun 28;8(1):60.  [Content Brief]

  [3]. Cong M, et al. MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination. Nat Cancer. 2020 Feb;1(2):222-234.  [Content Brief]