Demonstration of antibody specificity by antigen (peptide/protein) blocking
It is not uncommon to see more than 1 band in western when probed with a given antibody or see more diffuse staining in immunolocalization studies. The question arises which one of this band(s)/staining is specific. The antibody specificity is generally studied by competing with excess of antigen (peptide or protein) or immuno-neutralization with the antigens. In principle, a small volume of antibody (e.g., 1-5 ul) is first reacted with excess peptide (5-50 fold over the antibody; e.g., 1 ug antibody reacted with 5-50 ug peptide; exact amounts determined by titration) to neutralize it. The neutralized antibody can no longer subsequently bind to another antigen (a band of interest) or staining pattern. So the band(s)/stainign that is competed by the antigen/peptide is supposedly specific. If more than one band disappear by peptide/antigen competition then those bands have the antigenic determinants and could be considered either fragments of the large antigen or multimer.
A general protocol for peptide/antigen competition for an antibody
1. Determine the amount of antibody that is needed for 1 strip (e.g., 2 ml). For example, an antibody has given desired bands at 1:1000 dilution. So you will need 1 ul/ml antibody (2-ul antibody for 2-ml antibody solution). If an antibody were used at 1:5000 dilution then you would only need 0.2 ul/ml (use 2 ul of 1:10 dilution for better accuracy).
2. Take 2-ul antibody (or as needed) in 100 ul saline/PBS. Make 2 tubes. Add antigen/peptide solution (10-50 ug peptide or antigen added in 10-100 ul). Add same volume of saline/PBS (no peptide/antigen) to the other tube labeled as -peptide or No peptide. Mix gently.
3. Incubate both tubes at 37oC for 1-2 hrs and 2-24 hrs at 4oC.
4. Centrifuge the tubes for 15 min at 4oC in a microfuge (10-15000 rpm) to pellet any immune complexes. Carefully remove the supernatant. If no visible pellet is seen than just leave approx. 5-10 ul at the bottom to avoid disturbing invisible immune complexes. If you do not centrifuge the solution, it may give high background.
5. After centrifugation, make up the volume of supt. to 2 ml (or what is necessary depending upon the initial antibody taken) with buffer (PBS-Tween with or without BSA or milk or any buffer that was used initially). Use both antibodies (with and without antigen/peptide) for Western blotting or immunolocalization.
4. Observe the bands/staining that disappears.
Notes:
1. Higher the antibody titer or initial volume of antibody taken, higher will be the antigen/peptide necessary to completely block the antibody activity.
2. A partial inhibition of antibody activity is an indication that more antigen/peptide will be needed to completely block the antibody.
3. It may be necessary to optimize the amount of antigen/peptides by adding various amounts to a fixed concentration of antibody.