Hello,
I'm currently preparing a presentation on blotting for university and in one paper i've read it said:
"first, electrophoretic transfer of proteins from the SDS-PAGE gel to the filter is used instead of diffusion so that renaturation of proteins can occur during the transfer process, as the electric field rapidly removes SDS from the proteins; second, before the application of the probe, the filter is incubated with skim milk to block nonspecific and low-affinity binding sites"
I understand why it is done but how does it work and is there an alternative to skim milk ?
The skim milk is used to block nonspecific binding sites on the nitrocellulose or nylon members. Instead you can use BSA, Fetal calf serum, gelatin, BLOTTO, or any other protein which doesn't cross react with antigen, antibody or antibody-enzyme conjugate.
Hi. I agree with Shamsher. As the membrane is designed to bind proteins, antibodies would stick non-specifically to the membrane. To prevent this, the membrane is incubated with a protein which is not related to the antigen you are probing, eliminating undesired non-specific adsorption.
Skim milk is cheap and contains different proteins. So, there is a good chance for one of the milk proteins to bind to the membrane and block this non-specific binding site.
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