I want to use a particular antibody for IF. Some protocols say to dissolve the antibody in 10% FBS while other protocols say to dissolve it in 2% BSA. Can the BSA or FBS make a difference for the final staining? Is either FBS or BSA better?
Hello Charles,
As far as I know, both FBS and BSA will work as carrier proteins, thus they will probably function the same in your IF. Since BSA is very straightfoward and commonly used, I would try it first, unless you have good references recommending FBS.
By the way, I heard that FBS is very useful for blockage between antibody incubations, reducing the ammount of background fluorescence in the preparation. This may be a good reason to use FBS if you have previously tested the method.
Good luck
Either FBS or BSA can work. You may need to adjust the % of the FBS or BSA according to the background levels you observe.
Many people use normal serum from the host that the secondary antibodies were generated in. (If the secondary antibodies are raised in goat, then normal goat serum would be used as the carrier). My lab uses the same solution to pretreat to block non-specific labeling and to dilute the primary and secondary antibodies. Our standard blocker is 10% normal Goat serum + 5% BSA +1 fish gelatin+ 0.5% triton x-100 in HBSS or PBS used in combination with secondary antibodies raised in goat. If we're doing experiments that require a primary antibody raised in goat, then we substitute 2% normal donkey serum and use secondary antibodies raised in donkey. The BSA is a lyophilized, immunoglobulin-free fraction we purchase from Sigma. These blockers are also compatible with labeling using phalloidin or lectins (peanut agglutinin or wheat germ agglutinin, etc)
We only perform a second block step between the the primary and secondary antibody incubations if non-specifc background is exceptionally high.
Good luck
Hi Charles, The blocking buffer i use is
3% BSA
0.1% Gelatin
0.5% Triton X-100
0.05% Tween 20
0.05% Na Azide
PBS
I found in general serum and BSA both work well but my experience has been with donkey serum.
I use a very, very simple blocking buffer prepared fresh prior to each use consisting of 1% BSA in PBS containing 0.25% Triton X-100 (1 hour). I dilute my antibodies in 1% BSA in PBS for both primary and secondary. Primary is usually overnight at 4C, sometimes for 1-2 hours at RT, secondaries are 1 hour at RT.
Thanks for these answers, they were also usefull for me. But then I have an extra question, which BSA is the most indicated to IHC? there are different types and prices. Is there something that I should pay more attention when I purchase BSA?
thanks in advance
Hi there.
We usually use "BSA Fraction V" for blocking, both for certain antibodies in Western Blotting and IHC.
FBS and BSA make no difference in blocking non-specific binding when there is no 2nd Ab targeting to bovine IgG.
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