Blocking Peptide Competition Protocol
Description:
The Blocking Peptide Competition Assay (BPCA) utilizes the peptide used to generate the primary antibody to confirm the specificity of the Primary Antibodies binding affinity.
Reagent:
Antibody dilution buffer: 5% non-fat dry milk in 0.1 % PBST.
Procedure:
- Prior to performing the Blocking Peptide Competition Assay (BPCA), optimize conditions for Western blot. Optimized Western blot conditions should be replicated in the BPCA without change.
- This procedure describes the BPCA using the peptide and the corresponding primary antibody, where an antibody concentration of 1 μg/mL and a 200-fold molar excess of peptide are used in a total reaction volume of 2 mL. Molar ratios are based on the molar mass of IgG and the peptide as 150,000 Daltons and 1,500 Daltons, respectively.
- Transfer and immobilize the antigen on nitrocellulose or PVDF membrane as described above, preparing two identical test samples for analysis by BPCA. Incubate each membrane with Blocking solutions as described above.
- Prepare a 2 mL solution of primary antibody at a 2X concentration in dilution buffer relative to the optimum dilution used in the standard Western blot.
- Slowly warm the lyophilized peptides to room temperature, ideally under desiccation.
- Reconstitute each of the peptides to a concentration of 150 μg/ml using sterile PBS at room temperature. For a peptide with a molecular mass of 1500, reconstitution of 100 μg with 0.67 mL PBS yields a solution with a concentration of 150μg/ml.
- Allow the peptides to dissolve at room temperature, and then gently triturate several times using a pipette. Avoid introducing air bubbles.
- Label 2 test samples as follows:
a) Sample (a): water only. No peptide control.
b) Sample (b): peptide.
- Prepare 2X peptide stock solutions (4μg/ml) and water control by pipetting the following:
a) Sample (a): no peptide control: 973 μL dilution buffer plus 27 μL water.
b) Sample (b): peptide 2X stock: 973 μL dilution buffer plus 27 μL reconstituted (150 μg/ml) peptide.
- Pipette 1 mL of the 2X antibody stock into each of the samples marked (a) and (b). The tubes should be incubated for an hour at room temperature with gentle rocking.
Note: some antibody-peptide combinations require incubation for extended periods of time at alternative temperatures. If desired results are not obtained, try incubating the mixture for 1-2 hours at 37° C or for 2-24 hours at 4° C.
- Centrifuge the samples for 15 min at 4°C in a microfuge (10k-15k rpm) to pellet any immune complexes. Carefully remove the supernatant. If no visible pellet is seen then just leave approx. 5-10 μL at the bottom to avoid disturbing invisible immune complexes.
- The pre-incubated antibody in each of the two samples is ready for use. Pipette the contents of each sample onto the two identical test samples for immunoblotting (i.e. western blotting strips).
- Incubate each strip for 2 hours at room temperature, followed by several washes to remove unbound antibody.
- Transfer each strip to a new solution containing a labeled secondary antibody, prepared as described above.
- Remove unbound secondary antibody by thorough washing and develop bands following the standard protocol described above..
- The signals obtained with antibody incubated with sample (a) (water only, no peptide control) represent the expected signal. Signals obtained with sample (b) (peptide) are compared to the signal from sample (a) to determine if the peptide competes for antigen binding. A positive BPCA result shows no or little binding to a specific band for sample (b).
Note:
a) Ideally an excess of peptide is added to the antibody solution, where 200- to 500-fold more peptide is added on a molar basis relative to antibody. There does not appear to be any significant difference between a 200-fold and 500-fold excess. If the results of the BPCA are less than optimum, then try altering the concentration of peptide upward or downward (i.e. fix the antibody concentration) to improve the results of the BPCA. The best results are often determined empirically.
b) The higher the antibody titer or initial volume of antibody taken, the higher will be the antigen/peptide necessary to completely block the antibody activity.
c) A partial inhibition of antibody activity is an indication that more peptide will be needed to completely block the antibody.