BSA Blocking agent - How is work? (Sep/02/2009 )
May i know who understand how BSA working as blocking agent? What is the mechanism? Why it will able to block ELISA plate? It will able to block an empty ELISA plate surface?
Before using antibodies to detect proteins that have been dotted or transferred to a membrane, the remaining binding surface must be blocked to prevent the nonspecific binding of the antibodies. Otherwise, the antibodies or other detection reagents will bind to any remaining sites that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking. In practice, however, certain proteins perform better than others because they bind to the membrane or other immobilization surface more consistently or because they somehow stabilize the function of other system components. In fact, no single protein or mixture of proteins works best for all Western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system.
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.com/files/1601519%20WBhandbook.pdf
Hope this helps!!
BSA will block empty wells. Avoid surfactant in the buffer. Process for sticking is non-specific attachment.
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.
good luck
BSA can contain a lot of bovine Igs which can cause non-specific binding. You may want to look at an Ig-free BSA for your block
sgt4boston on Wed Sep 2 20:20:46 2009 said:
BSA will block empty wells. Avoid surfactant in the buffer. Process for sticking is non-specific attachment.
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.
good luck
AVEA on Wed Sep 2 16:50:42 2009 said:
Before using antibodies to detect proteins that have been dotted or transferred to a membrane, the remaining binding surface must be blocked to prevent the nonspecific binding of the antibodies. Otherwise, the antibodies or other detection reagents will bind to any remaining sites that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking. In practice, however, certain proteins perform better than others because they bind to the membrane or other immobilization surface more consistently or because they somehow stabilize the function of other system components. In fact, no single protein or mixture of proteins works best for all Western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system.
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.com/files/1601519%20WBhandbook.pdf
Hope this helps!!
Hi, guys,
What if the substrate of the well is metal, say aluminum, gold, or platinum? What that affect the blocking when using agents like BSA or Buffer B? Thank you so much! I am an Electrical Engineering majored student, so most of our "biomedical" experiments are done on the metal surface or substrate like electrodes.
Cheng Cheng on Tue Sep 24 15:31:04 2013 said:
This is a difficult one, your best bet will be to do some empirical studies using different protein solutions and concentrations to see what works. Some proteins do bind well to metals, but I have no idea which ones.Hi, guys,
What if the substrate of the well is metal, say aluminum, gold, or platinum? What that affect the blocking when using agents like BSA or Buffer B? Thank you so much! I am an Electrical Engineering majored student, so most of our "biomedical" experiments are done on the metal surface or substrate like electrodes.