Hi,
I want to use an Affymetrix kit for EMSA (AY1000) with biotin-labeled probes, but the blocking and wash buffers are already empty. Do you know the components of the buffers? I found so many different recipes, which make me confuse, because some contain BSA, SDS etc and others not!
I recently ran into the same dilemma with the ThermoSci Biotin Detection kit, and had success using
Tris-buffered saline (TBS), 2L
20 mM Tris-HCl, 500 mM NaCl (pH 7.5)
Adjust pH to 7.5 with HCl. Adjust volume to 2 L with diH2O.
and for Blocking
3% BSA-TBS
Add 1.0 g BSA to 100 ml TBS; stir to dissolve.
Kristina,
Since the biotin-labelled EMSA kit required the 'physiologically correct shape' of avidin you can use (almost) any physiological buffer to wash with (TBS, PBS... etc).
For blocking, you can also use (almost) any blocking solution for Western blots. I would not use non-fat dried milk though, but bovine serum albumin, ovalbumin, gelatin... should all work.
If you're having high background problems, add 0.1% (v/v) of Tween-20 (or Triton X-100, NP-40...etc) to your wash and block solutions.
Lorenzo
Thanks for your answers. I was not sure about using the same as for western blot.
I will try TBS and BSA. :)
Hi Krisitina,
I found a handy reference for these buffers. I'll attach it. Hopefully what you are looking for is on it. I'm not sure which binding buffer you are asking about, so don't want to just list the recipe and end up stearing you in the wrong direction!
I mean the binding buffer for the reaction mix with the labeled TF and my protein of interest. I found this recipe for 10 ml
0.5 ml of 1 M Tris HCl (pH 8.0)
3 ml of 2.5 M KCl
50 μl of 0.5 M EDTA (pH 8.0)
50 μl Triton-X 100
7.87 g glycerol
add DTT fresh before use
Any suggestions?
Hi. Were you successful using the TBS-BSA as the blocking buffer? Thanks.
I've used this protocol with some success
http://www.protocol-online.org/prot/Protocols/Aimee-s-non-Radioactive-EMSA-Protocol-3455.html
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