What is the composition of TBS, TBST and transfer buffer to perform semi-dry western blot?
Hi,
We use Bjerrum Schafer-Nielsen with SDS for our semi-dry WB transfers.
Please see the following document for various transfer buffer recipes, including the one we use:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6211.pdf
We use PBS for our washing steps (we only add detergent if we end up with a background problem, which is rare).
Here are recipes:
TBS 10X
24.2 g Tris
88 g NaCl
Adjust the pH to 7.6 with HCl.
Complete to 1 liter with distilled water.
TBST
To 1 liter of TBS 1X, add 1 mL of Tween 20
PBS 10X
40 g NaCl
1 g KCl
7.2 g Na2HPO4-7H2O
1.2 g KH2PO4
Adjust the pH at 7.4 with HCl.
Complete to 500 mL with distilled water
Hope this helps!
Thank You for your kind reply. I have tried this protocol and it is working nicely.
Hi,
What is the difference between washing the membrane with TBS vs PBS? Is there a clear and noticeable difference?
Thank You!
I haven't found much of a difference. However, when probing for phosphorylated proteins many labs prefer TBS because it lacks phosphates, which some complain interferes with the antibody. Our lab has successfully probed for phosphorylated antibodies using PBS buffer, so this issue seems to be possibly antibody-specific.
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