實驗概要
The peptide competition assay/blocking procedure can be used in western blotting, IHC and ELISA. The peptide competition assay (PCA) is a recommended procedure for confirming the specific band reactivity of an antibody, especially domain specific antibodies like phospho-specific antibodies. It is not uncommon to see more than one band on immunoblots of lysates when probing with a primary antibody. The PCA provides a means of determining which band or staining pattern is specific for the antibody. The experiment should be run in parallel; with the peptide immunogen (e.g. phosphorylated form peptide) and with or without the irrelevant peptide (e.g. non-phosphorylated peptide).
實驗步驟
In the PCA, the antibody is pre-incubated with the peptide prior to use in immunoblotting assays. The immunoblotting experiment is run in duplicate; one with antibody pre-incubated with peptide and another one with a control antibody not pre-incubated with peptide. All other parameters of the immunoblotting experiment should remain constant i.e. the same dilutions and buffer conditions throughout the experiment.
The following example uses phosphorylated and non-phosphorylated peptides with phospho-specific primary antibody. The primary antibody concentration is used at 1 μg/ml with a 200-fold molar excess of peptide.
Molar ratios are based on the molar mass of IgG as 150 000 Daltons. Based on weight, the primary antibody can be incubated with a five-fold excess of peptide.
We recommend first optimizing conditions for immunoblotting for both the antibody and antigen, including the amount of lysate, dilution of primary and secondary antibodies, dilution buffer, and blocking buffer.
1. Transfer and immobilize the antigen on nitrocellulose or PVDF membrane and prepare 3 identical test samples for analysis by PCA.
2. Prepare a solution of primary antibody at a 2X concentration in dilution buffer.
Note: 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
3. We usually recommend storing each peptide stock at concentration of 100 μM (the phosphorylated and non-phosphorylated forms of the immunizing peptide) in the same dilution buffer as the primary antibody, at room temperature.
Note: Refer to the molecular weight of the peptide for the exact amount and volume.
For a peptide with a molecular mass of 1500, reconstitution of 100 μg
with 0.67 ml water yields a solution with a concentration of 100 μM
4. Label 3 antibody test samples as follows:
1) Sample (a): dilution buffer only. No peptide control.
2) Sample (b): phosphorylated peptide.
3) Sample (c): non-phosphorylated peptide.
5. Prepare 2X peptide solutions (2.66 μM) and the three samples as following:
1) Sample (a): dilution buffer only. No peptide control.
2) Sample (b): phosphorylated peptide 2X solution.
3) Sample (c): non-phosphorylated peptide 2X solution.
6. Mix the related volume of the 2X peptide solutions into each antibody sample marked (a), (b) and (c) in glass test tube in order to prevent the coating of the antibody. The tubes should be incubated for 30 min at room temperature with gentle rocking.