Antibodies, also known as Immunoglobulins (Ig), are produced and secreted by plasma cells, which originate from the proliferation and differentiation of B cells. They exhibit remarkable specificity in recognizing and binding to antigens. Antibodies can be classified into five classes based on the Fc region of their heavy chains: α-IgA, δ-IgD, ε-IgE, γ-IgG, and μ-IgM. The light chains are of two types:λ and κ (Figure 1).

In fact, the antibodies we commonly refer to are primarily the first antibodies, which are proteins capable of specifically binding to antigens. These include monoclonal antibodies (mAb) and polyclonal antibodies (pAb). Monoclonal antibodies can recognize a single epitope on an antigen, while polyclonal antibodies are capable of specifically binding to different binding sites on the same target antigen (Figure 2).

Although both are antibodies, monoclonal antibodies (mAb) and polyclonal antibodies (pAb) exhibit numerous differences (Table 1).

The secondary antibody, also known as the antibody to the antibody, is capable of binding to the primary antibody. Its primary function is to detect the presence of the primary antibody and amplify its signal. While the primary antibody can specifically bind to the target protein, it often requires labeling with a luminescent substance or a chromogenic group to be detectable. Therefore, a labeled secondary antibody (e.g., with fluorescence, radioactivity, chemiluminescence, or a chromogenic group) that can be detected is used to bind to the primary antibody. In this way, when the primary antibody binds to the substrate, it can be detected through the secondary antibody.

Selection of Primary Antibody

NO.1 Analyze Application Types：

Select antibodies based on the type of experiment conducted.
The "Application" section on the product page information on the MCE official website lists the applicable experimental types for which the antibody has been experimentally verified.
For example, Application: WB, ICC/IF, IHC-P, FC

NO.2 Analyze Sample Species：

Select antibodies based on the species of the sample.
The "Reactivity" section on the product page information on the MCE official website lists the species information for which the antibody has been experimentally verified.
For example, Reactivity: Human, Mouse, Rat

NO.3 Select the Host Species of the Antibody

Generally speaking, when using a secondary antibody coupled to bind with an unconjugated primary antibody, the choice of the species of the host animal for the primary antibody is crucial.
① To avoid cross-reactivity between the secondary anti-immunoglobulin antibody and endogenous immunoglobulins in the sample, the source organism of the selected primary antibody should be different from that of the sample. For example, if studying mouse proteins, a primary antibody produced by an organism other than mouse should be selected. Assuming a rabbit-derived primary antibody is chosen, the secondary antibody can be selected as anti-rabbit IgG coupled with a detection molecule (enzyme, fluorescein, biotin, etc.).
② If a conjugated primary antibody is chosen, the above situation does not apply. For detection methods other than immunohistochemistry, which involve samples without endogenous immunoglobulins, the host species of the antibody has little impact.
The "Host" section on the product page information on the MCE official website lists the host source of the antibody.
For example, Host: Rabbit

NO.4 Analyzing Structural Domains of the Target Protein

① Target Protein Region for Testing: Verify that the immunogenic region (full-length protein, protein fragment, peptide, whole organism, or cell) is consistent with or contained within the protein region to be detected.
② Sample Extraction or Processing: Some antibodies require samples to be processed in specific ways. Many antibodies can only recognize protein that have been reduced and denatured (which exposes epitopes hidden within the secondary or tertiary structures formed by protein folding). On the other hand, some antibodies can only recognize epitopes on protein in natural and folded state.

Selection of Secondary Antibody

NO.1 Hosts Origin of the Primary Antibody

Choose the corresponding secondary antibody against the host of the primary antibody based on its host origin.

① If the primary antibody is a mouse-derived monoclonal antibody, the secondary antibody should be chosen as an anti-mouse secondary antibody (e.g., goat anti-mouse or rabbit anti-mouse).
② In special experiments, such as double labeling experiments, if the primary antibodies are both rabbit-derived and mouse-derived, the corresponding secondary antibodies should simultaneously include both anti-rabbit and anti-mouse.

NO.2 Conjugation Labels of the Secondary Antibody

The main types of conjugation labels for secondary antibodies include:

① Enzymatic labels (e.g., AP, HRP, ...)
② Fluorescent labels (e.g., FITC, PE, Cy7, ...)
③ Other labels (e.g., Biotin, ...)
The choice of the probe for the secondary antibody mainly depends on the specific experiment.
For example, in Western blot and ELISA, commonly used secondary antibodies are enzyme-conjugated. In cell or tissue labeling experiments (such as immunohistochemistry, immunocytochemistry, and flow cytometry), secondary antibodies labeled with fluorescent groups are typically used. In immunohistochemistry, secondary antibodies labeled with horseradish peroxidase (HRP) or alkaline phosphatase (AP) can also be employed.

Selection of Loading Controls Antibody

After selecting antibodies, it's crucial to carefully consider how to choose a loading controls. It's a common mistake to blindly choose "GAPDH" or "β-Actin" regardless of the sample type. Furthermore, the selection of loading controls should also be based on the corresponding expression levels of the loading controls proteins in the samples.

1. Do you really know your protein?

Does your protein easily undergo modifications? Does it tend to form aggregates? Are there any isoforms? Even the species origin can affect the molecular weight of a protein. Here, Little M recommends the "UniProt" website.

2. Has your sample been completely lysed?

Here, Little M suggests choosing a suitable lysis buffer, such as RIPA Lysis Buffer (Strong) , and fully lysing the sample through homogenization or sonication.

3. Have you really chosen the right antibody?

Dear researchers, remember to choose antibodies that have been validated and are highly specific. Here, Little M can't help but brag a little - the antibodies provided by Little M all meet strict and controllable quality standards.

4. Are your experimental operations truly flawless?

Here, Little M offers a few suggestions regarding experimental operations: Pay attention to thoroughly mixing the gel solution and pay close attention to the electrophoresis conditions.
1. Choose suitable blocking solution (HY-K1027 ), antibody diluent, and membrane washing solution (HY-K1022 , HY-K1025 ).
2. During the gel preparation process, pay attention to the state of the gel. During the experiment, select appropriate electrophoresis conditions based on your target protein.

MCE offers 4000+ primary antibodies and various secondary antibodies, covering popular targets, to help your scientific research achieve "success in all directions"! Come and check out our star products (due to space limitations, only 6 star representatives are invited):