I am developing a Lateral Flow Assay and need to block a nitrocellulose membrane with BSA for my initial sample test. How do I appropriately coat my membrane with BSA to avoid non-specific binding? I am using 10% BSA. The antibodies and antigens are very low in molecular weight (20-30 kDA). I will also be diluting the antigen/protein that I will be testing for in water. One of my sample tests will also contain neutravidin immobilized on the membrane for my test line and an anti-rabbit goat antibody on my control line. My conjugate will also consist of carbon nanoparticles and an antibody (low weight). On the other sample test, I will be immobilizing an antigen on the test line and the same secondary antibody as the other sample test on my control line. The conjugate remains unchanged.
10% (w/v) solution of high-quality BSA is useful for saturating excess protein-binding sites on membranes and microplates in immunoassays. Blocker BSA Buffers are most frequently diluted 10-fold (to 1% BSA) in 1X Tris-buffered saline for initial testing, but other buffer concentrations can be beneficial for specific systems. Blocker BSA is usually more effective than nonfat milk blocking buffers for biotin-avidin systems because it contains a single purified protein that is devoid endogenous biotin.
Although the blocking agents are used in sample and conjugate pads, however, to remedy non-specific bindings, blocking of the NC membrane after Ab printing is essential but its really a trial and error work. A washing step is usually required after blocking.
Many reagents are available from proteins such as BSA, non-fat dry milk and casein to some hydroxyl-containing compounds such as PVA and PEG. Prepare them in proper buffers such as PBS and sucrose and immerse the membrane in blocking solution followed by washing step. Note that usually low concentrations of compounds are used in LFIAs.
Hope this information being useful!
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