As many of you already know and we have commented on other occasions on the blog, the Western Blot technique is very useful since it combines the resolution capacity of polyacrylamide gel electrophoresis (PAGE), the specificity of the antibodies and the sensitivity of enzyme tests. Although the entire process itself must be done with great care, the key factor in this process is the ability of the primary antibody to detect the protein of interest.
Theoretically, the antibody should bind only to the protein it is targeted for, and therefore the thickness of the band should correspond to the amount of protein present in our experiment. However, many times, more bands appear above or below our protein of interest, which makes quantification more complicated.
In our blog, we have made some posts where we talked about how to validate antibodies, but in this case, we will focus on how to validate a monoclonal or polyclonal antibody for transfer in Western Blot.
To do this you have to follow several steps:
- CHOOSE A PRIMARY ANTIBODY:
The precision of the Western Blot results depends largely on the quality of the primary antibody used.
The choice of the primary antibody depends on several factors:
The antigen we want to detect.
The folding of the protein of interest, since, depending on how it is folded, the epitopes that are exposed vary.
In this case, both monoclonal and polyclonal antibodies work very well.
This antibody must be specific, sensitive and reproducible enough to detect the target protein, using the lowest concentration of it. High concentrations can give rise to unspecific bands or excessive signal intensities, which will prevent us from efficiently quantifying this protein.
- VALIDATE A MONOCLONAL OR POLYCLONAL ANTIBODY:
Antibody validation is a process in which it is demonstrated, by means of specific laboratory techniques, that the performance characteristics of an analytical method are adequate for its intended use. So it is a process in which primary antibodies are analyzed to determine their sensitivity, specificity and reproducibility in the context in which they are to be used.
According to the International Antibody Validation Working Group (IWGAV), several techniques can be performed to validate antibodies intended for Western Blotting:
Recombinant expression validation
Validation of independent antibodies
MS capture validation
GENETIC VALIDATION: SIRNA LEVELS
To compare staining intensities of the antibodies with the data of an RNA sequencer from the same samples on different tissues or cells with variable expressions of our protein of interest. The antibody is considered to be specific when its signal coincides with the RNA levels of the samples analyzed.
GENETIC VALIDATION: KO MODELS
They function as a negative control. Our sample is compared with a negative sample from a tissue that does not express the protein of interest, in cells or knockout animals (CRISPR) or can also be done in genetically ablated animals (RNAip).
INDEPENDENT ANTIBODY VALIDATION: IHC AND ICC
This method is based on comparing staining levels using two independent antibodies without overlapping epitopes. For this, the protein is studied in tissues (IHC) or cells (ICC) that express the protein of interest at different levels.
Antibodies would have to be compared in a set of relevant tissues. If they generate a similar staining pattern, these validate each other, since the antibody recognizes the correct protein based on cell location.
RECOMBINANT EXPRESSION VALIDATION: CELL TREATMENT
Level of protein expression manipulated within cells.
For example, the target protein could be overexpressed in a cell line that does not express the protein of interest. An evolution of the antibody staining will be carried out by analyzing samples of cell lysates with and without recombinant expression of the target protein. Thus, if a band does not appear or the band is very weak, it will correspond to the lysate of the unmodified cell line; and a strong band in the cell line with recombinant expression.
ORTHOGONAL VALIDATION: MASS SPECTROMETRY
The staining pattern and the size of the protein detected by the same antibody would have to be compared by mass spectrometric (MS) capture.
Antibodies are considered enhanced when staining intensity and protein expression levels show the same pattern, which will mean that the antibody is directly interacting with the target protein.
For this method it is recommended to use at least two samples of cells or tissues, so that the protein of interest expresses the target at different levels.
MS CAPTURE VALIDATION
In this case, the molecular weight of the antibody band and the size of the protein obtained by MS capture are compared, in which the electrophoresis gel is cut and each sample is analyzed separately by proteomics. The proteins in each cut of the gel are digested to form peptides.
The antibody related band should be equivalent to the predicted target protein and its peptide (s).
It is important to remember that these validation data should always be included in the results of your experiment together with the references indicating where the actual antibody described has been used.