Pre-incubation of antibody with the immunogen to demonstrate specificity
Nonspecific binding of antibody to proteins other than antigen, may sometimes occur. It is generally more frequently with polyclonal antibodies, but may occur with a monoclonal antibody as well.
To determine which band or staining is specific to the immunized peptide blocking experiments can be performed. Before proceeding to the staining protocol, the antibody was neutralized (incubated with an excess of a peptide corresponding to the epitope recognized by the antibody). An antibody that binds to the blocking peptide is no longer able to bind to an epitope present in a protein on Western Blot or in the cell. In neutralizing antibody was then used alongside antibody alone, and the results compared. By comparing the color of blocking antibodies against the antibody alone, you can see which color is specific: this color will be absent from the Western Blot or immuno performed with neutralizing antibody.
Materials and reagents
Blocking buffer (typically TBST plus 5% nonfat dry milk, or 3% BSA in Western Blot, PBS or plus 1% BSA for IHC)
Blocking antibodies (immunizing) peptide
Two identical samples (e.g., Western Blot with two identical panels, cut in half, two slides containing the cells of interest, etc.)
Determination of the optimal concentration of antibodies that consistently gave positive results in particular its Protocol. The use of this concentration, to determine how much antibody would have for the two experiments. For example, the antibody was successfully used in the Western Blot of 0.5 g / ml. You will have 2 ml solution of the antibody to stain a strip of Western Blot. Thus, you can use one mu antibody in 2 ml buffer for each band.
Dilute the required amount of antibody in Blocking buffer to a final volume required for both experiments. Divide this equally into two tubes. The first tube marked “locked” blocking peptide added to a final concentration of 1 ug / ml (2 ug total peptide in this example). The second tube labeled “Control” is added an equivalent amount of buffer.
Incubate for two tubes, with stirring at room temperature for 30 minutes, or overnight at 4 ° C.
Perform protocol coloring two absolutely identical samples using a blocking antibody and monitored for one another. Be careful not to mix the two bands using antibodies blocked and control!
Observe the color. Coloring which vanishes when using blocking antibody is specific for the antibody. (See note below)
1. If more than one band disappears in Western Blot of competition peptide / antigen, these groups contain antigenic determinants and can be fragments of full antigen or a complex comprising an antigen.