BSA Blocking agent
Posted 01 September 2009 - 11:37 PM
Posted 02 September 2009 - 08:50 AM
Blocking Nonspecific Sites
The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal to noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding.
The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection label used. For example, in applications where alkaline phosphatase conjugates are used, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Many factors, including various protein:protein interactions unique to a given set of immunoassay reagents, can influence nonspecific binding. The most important parameter when selecting a blocker is the signal:noise ratio, measured as the signal obtained with a sample containing the target analyte, as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background staining and a reduced signal:noise ratio. Using excessive concentrations of blocker may mask antibody:antigen interactions or inhibit the marker enzyme, again causing a reduction of the signal:noise ratio. When developing any new immunoassay, it is important to test several different blockers for the highest signal:noise ratio in the assay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics.
This might not say how BSA works but does give information on how choose blocking buffers and what to look for to see if one is working poperly. I was also able to find a good Western Blotting Technical Guide for you at http://www.piercenet... WBhandbook.pdf
Hope this helps!!
Posted 02 September 2009 - 12:20 PM
Depending upon what you are trying to do, for elisa BSA is pretty standard. However, depending upon the assay samples etc etc there are many other blockers out there from milk proteins, artificial blockers and finally fish proteins. BSA is a good starting point.
You can never over-block. Just make sure your solution does not evaporate during this stage. People do RT for 30 min or 2 hrs or overnight 4C. (be sure to seal your plates).
Others leave the solution in with plate sealed with parafilm until needed.
If you wish to dry your plates block with mixture of BSA and sucrose. Decant and dry inverted overnight, store 4C.
Posted 02 September 2009 - 05:14 PM