re: biotin blocking
|From:||Laurie Brett <firstname.lastname@example.org>|
To Barb. Terret
Regarding your question on biotin blocking reagents.
Since we treat most of our workload with pressure-cook antigen retrieval, we
have a lot of potential endogenous biotin binding. So we block everything
for endogenous biotin. To use commercial kits would cost quite a lot (we do
around 18,000 immunos a year) so we make our own solutions:
1. 10% aqueous eggwhite 30 mins. (bought as a dried powder from Sigma). Mix
well until most of it is dissolved then centrifuge briefly and use the
supernatant (removes heavy particulates). This is the Avidin solution.
2. Rinse in PBS (or TBS).
3. Dilute all primary antibodies in a solution containing 10mg d-biotin
(also from Sigma) per 100mLs PBS (you can include your other usual blocking
agents such as normal serum or BSA). Incubate in primary for at least
The eggwhite also acts as a good all round blocking agent and can be used in
place of normal serum. It doesn't require any buffer - natural biological
buffering keeps it at about about pH7.2 and it is very cheap compared with
Avidin. We find it works as well as commercially supplied kits.
Senior Biomedical Scientist,
Cellular Pathology Department
Western General Hospital
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