The protocol of the antibody from NOVUS (NBP1-21502) recommends to use the mixture of 1%BSA and 5%non-fat milk as the blocking buffer for Western blot. I am curious the principles and reasons to use BSA and non-fat milk AT THE SAME TIME. Isn't it to use one of them for blocking in this antibody?
ref: https://www.novusbio.com/products/ferroportin-slc40a1-antibody_nbp1-21502
Thanks
Hi Jaspreet,
blocking protocols are usually optimized using different blocking agents. Finally the best blocking protocol is used for the assay. There is often no ratinale but a lot of experience driving the development.
Sometimes it is useful to have a larger protein (BSA) combined with smaller proteins like casein and alpha/beta lactoglobulins (major proteins of milk powder) to block all protein binding surfaces in your assay. All these proteins are cheap, available in large quantities and show a low binding to other proteins. So when they bind to the hydrophobic surface they will denature an stick with their hydrophobic internal parts to the surface whereas the hydrophilic outer parts of the blocking proteins are presentetd to the assay reagents later. This results in a protein friendly and low binding surface which prevents assay proteins (Analyte, antibodies, enzymes) from unspecific binding and sticking to any surface in the assay resulting in high background or unspecific signals in the assay later.
I hope this helps a bit to understand how blocking works and why blocking protocols can be so different .
Best regards Dieter
Hi there,
Yes we can use anyone of the blocking buffer at a time, in our lab we generally use 5% skim milk as a blocking buffer for all the antibodies and it works well. Note: we have antibodies from Novus, so I think you can go with 5% skim milk. However in case of phosphorylated proteins it is recommended to use 3% BSA as a blocking buffer.
Hi Jaspreet,
blocking protocols are usually optimized using different blocking agents. Finally the best blocking protocol is used for the assay. There is often no ratinale but a lot of experience driving the development.
Sometimes it is useful to have a larger protein (BSA) combined with smaller proteins like casein and alpha/beta lactoglobulins (major proteins of milk powder) to block all protein binding surfaces in your assay. All these proteins are cheap, available in large quantities and show a low binding to other proteins. So when they bind to the hydrophobic surface they will denature an stick with their hydrophobic internal parts to the surface whereas the hydrophilic outer parts of the blocking proteins are presentetd to the assay reagents later. This results in a protein friendly and low binding surface which prevents assay proteins (Analyte, antibodies, enzymes) from unspecific binding and sticking to any surface in the assay resulting in high background or unspecific signals in the assay later.
I hope this helps a bit to understand how blocking works and why blocking protocols can be so different .
Best regards Dieter
Dear Lo Tzu Yu,
as nobody knows which interaction causes unspecific signals in an assay it is quite common to test different blocking agents during assay development. Sometimes it helps to combine a large blocking protein ( BSA abot 66 kD) and a mixture of smaller proteins from milk ( casein, lactoglobulin, etc.). In principle it is a trail and error development based on personal experience or lab knowledge. I am sure that NOVUS optimized the protocol - but nevertheless sometimes you might have to do your own optimization eg when you use different membranes or blotting buffers / protocols.
Good luck and best regards
Dieter
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