Why milk is used in Western blotting? - (Oct/05/2006 )
Tween is a non-ionic detergent used for breaking unwanted lipid-protein interactions in order to improve the background.
-spanishflower-
u mean that fats may block antibody binding, but antibodies are supposed to bind to the proteins , not to milk.....
other suggestions...
-strawberry-
-strawberry-
I think that lipids can bind proteins, and both antigen and antibody are proteins, and if lipids bind to them, this will make more difficult the specific bindings antigen-Ab.
-pumuki-
may be you'r right ...pumuki
-strawberry-
QUOTE (strawberry @ Oct 14 2006, 04:11 PM)
one more Qn: how milk is applied in WB, i mean how to prepare it?
you may buy dried skim milk form a biotech company, or much more cheaper, from a health food store; anyway, you dilute in your preferred buffer system for immunoblots (TBS, TTBS, PBS), stir it, and do not forget to filter; concentration is somehow philosophy and experience, it may range from 0.5 to 5 %; so optimize it for your system
-The Bearer-
And how about BSA concentration? Is it 3% or 5%?
-saya-
QUOTE (saya @ Nov 1 2006, 07:05 AM)
And how about BSA concentration? Is it 3% or 5%?
i use 3%BSA in 1xTBST
-spanishflower-
QUOTE (spanishflower @ Oct 31 2006, 08:14 PM)
QUOTE (saya @ Nov 1 2006, 07:05 AM)
And how about BSA concentration? Is it 3% or 5%?
i use 3%BSA in 1xTBST
Thanks Spanishflower
. -saya-
QUOTE (strawberry @ Oct 14 2006, 08:11 AM)
one more Qn: how milk is applied in WB, i mean how to prepare it?
Usually in TBS solution with Tween20 (detergent), but PBS is also used. the concentration may range from 3-10%. Be sure what pH you will use and adjust AFTER milk solution preparation. For comercial ABs, the company usualy provides a protocol for optimal conditions to blocking buffer, time incubations, AB dilution solution, etc. If you are testing an AB, you will need to test different concentrations, pH and buffers. Godspeed
-Robertzar-