Selection guide for WB products!

1.When should I choose to use the WB method to detect protein?

According to different sample types and testing purposes, different types of testing methods are selected. The sample types tested by WB are mainly cells or tissues, which are characterized by qualitative and semi-quantitative.

2.How to choose the right product for WB experiment?

a. How to choose the right primary antibody?

1）Species of experimental samples：Such as human, rat, mouse, pig, goat, etc. Antibodies of the same species or cross-reactive should be selected. Antibodies may cross-react with the same target protein of different species;

2）Experimental type：WB，Elisa，IHC，ICC，FACS；

3）The structure of the sample protein：Full-length protein, protein fragments, peptides, etc.；

4）Monopolyclonal antibody：Monoclonal: strong specificity, relatively small affinity; polyclonal: weak specificity, strong affinity, select monoclonal or polyclonal antibodies according to specific experiments；

b. How to choose the right secondary antibody?

1）Host species of primary antibody：Select the corresponding second antibody according to the source of the first antibody. If the first antibody is the source of the mouse, the second antibody can be used as the anti-mouse antibody.

2）Labelled substance：The probes coupled to the secondary antibody mainly include enzymes (horseradish peroxidase HRP and alkaline phosphatase AP, etc.), fluorescent groups (FITC, PE) and biotin. Which probe's secondary antibody to choose depends mainly on the specific experiment. The commonly used secondary antibody for western blot is an enzyme-labeled secondary antibody;

c. How to choose a suitable internal reference?

1）Species source: corresponding to the species source of the sample, such as mammals or plants;

2）Cell location: select according to the cell location of the target protein;

3）Molecular weight: at least 5 kDa difference from the molecular weight of the target protein;

4）Expression factors: Because GAPDH is an enzyme in the glycolysis process, GAPDH cannot be selected as an internal reference for studies on sugar metabolism (such as diabetes models); and in some cells, hypoxia can also affect the expression of GAPDH, and this is not the case. You can choose GAPDH as an internal reference.

3.Common problem

a. Can the antibody be used in other experiments (such as IP, IF, ELISA, FCM)?

There is a certain correlation between different applications: those who can do IHC are more likely to do IF, and those who can do WB are generally verified by ELISA; general antibody instructions list which types of analysis the antibody has been verified to be suitable for, such as: it can be applied to WB, IHC, ICC, ELISA, FCM analysis, etc. If the antibody specification does not mention the type of application, it does not mean that the antibody is not suitable for this type of analytical application, but only that it has not been verified by such analytical tests.

b. Can it be applied to the WB detection of XXX samples?

From the perspective of homology of different species; general antibody instructions list the specimens of which animal or human the antibody has been verified by experiments, such as: it can be used for rat, mouse, human, pig, goat and other animal specimens experiment of. That is to say, antibodies with the same species or cross-reactivity should be selected. Antibodies may cross-react with the same target protein of different species because of the high amino acid sequence homology;

If the type of the sample is not included in the cross-reaction species list in the antibody manual, it does not mean that the antibody is not suitable for detecting the protein of the species, but that the species has not been verified by this antibody test.

c. Does the antibody contain XXX ingredients?

Although BSA, glycerol and sodium azide are essential ingredients to maintain antibody stability and prevent antibody contamination, these components can also hinder the effective coupling of antibodies with fluorescent dyes, metals and enzymes；

BSA competitively binds the marker with the first antibody, thus greatly reducing the coupling efficiency of the antibody with fluorescent dyes, metal ions, isotopes or enzymes；

Sodium azide in antibody solution is cytotoxic, which limits the use of antibodies in cell culture, and is not suitable for staining or treatment of living cells, or for in vivo research. In addition, sodium azide can interfere with antibody coupling and inhibit the activity of horseradish peroxidase (HRP).

d. Is there any difference between milk and BSA closure?

Antibodies may be sensitive to sealants. In general, BSA produces clearer results because BSA contains fewer proteins, so antibodies are less likely to cross-react with it；

Milk contains more kinds of blocking proteins, which can block more different types of proteins. It is not recommended for the study of phosphorylated proteins; casein contained in milk is a phosphorylated protein that may lead to non-specific binding of antibodies, forming a high background.

4.Product recommendation

Cell and protein research tools

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