Pre-adsorption, also known as antibody blocking or neutralization, is a crucial step in antibody protocols like Western Blot (WB), Immunocytochemistry (ICC), and Immunohistochemistry (IHC) to reduce non-specific binding. It involves incubating an antibody solution with the antigen or a closely related peptide before applying it to the sample.
The process typically involves the following steps:
Steps for Antibody Pre-adsorption
Based on the standard protocol, pre-adsorbing antibodies helps improve specificity by allowing antibodies that bind non-specifically to related targets to be captured before they encounter your actual sample.
Here's a breakdown of the process:
- Optimize Antibody Concentration: Determine the optimal working concentration of your primary antibody for your specific application (WB, ICC, or IHC) in the appropriate buffer. This is the concentration you would normally use without pre-adsorption.
- Prepare Stock Solution: Prepare enough of this concentration-optimized antibody solution for at least two separate experiments or uses.
- Divide the Antibody: Equally divide the prepared antibody solution into two separate tubes. One tube will serve as your control (unadsorbed antibody), and the other will be used for pre-adsorption.
- Add Blocking Agent: To the tube designated for pre-adsorption, add an excess amount of the blocking peptide or protein specific to your antibody. The reference suggests adding 2 to 5 times excess by weight of the peptide or protein compared to the antibody weight.
- Note: The exact amount of peptide/protein needed can vary and may require optimization. Using an excess ensures that the antibodies that bind to the target epitope (or similar epitopes) are saturated by the blocking agent.
- Incubation: Incubate the antibody-peptide/protein mixture. The incubation time and temperature can vary depending on the specific antibody and protocol, but common conditions include incubating at 4°C overnight or at room temperature for 1-2 hours with gentle mixing. This allows sufficient time for the antibody to bind to the blocking agent.
- Separation (Optional but Recommended): After incubation, it's often beneficial to separate the antibody-peptide complexes from the free antibodies that are still intended to bind to your target. This can be done by centrifugation to pellet any aggregated complexes, or sometimes the supernatant is simply used. The unadsorbed control antibody should undergo the same incubation and centrifugation steps without the blocking agent added.
- Use the Supernatant: Carefully collect the supernatant containing the pre-adsorbed antibody (the free antibodies remaining after incubation with the blocking agent). Use this supernatant as your primary antibody solution for your experiment (WB, ICC, or IHC).
Comparing the results obtained with the pre-adsorbed antibody supernatant versus the unadsorbed control antibody solution helps verify the specificity of your antibody and identify bands or staining that are likely due to non-specific binding.
Why Pre-adsorb?
Pre-adsorption is typically performed when an antibody shows unwanted cross-reactivity with targets other than the primary target, especially in complex samples like cell lysates or tissue sections. By incubating the antibody with the blocking peptide (which mimics the epitope) or a protein containing the epitope, you effectively "soak up" the antibodies that would otherwise bind to your sample.
Practical Considerations
- Blocking Agent Choice: Ensure the blocking peptide or protein is highly pure and corresponds to the specific epitope the antibody was raised against.
- Optimization: The optimal ratio of blocking agent to antibody often requires empirical testing.
- Control: Always run an unadsorbed control alongside the pre-adsorbed sample to properly assess the effect of the pre-adsorption step.
This technique is particularly valuable when working with antibodies that recognize highly conserved epitopes or targets that are present in multiple isoforms or protein family members.
