SDF-1(CXCL12) western blot - (Jun/02/2009 )
mdfenko on Feb 24 2010, 09:24 AM said:
you can avoid this by using fresh reducing agent or eliminating reducing agent from the sample (you can get away with this with some but not all proteins).
there are some potential methods to remove the artifact from the sample but we have had no luck with them (we gave a half-hearted try then just eliminated the reducing agent when we needed to eliminate the band).
Can you send me a protocol you have tried???
What exactly do you mean with "eliminating reducing agent"; just donīt add to your lysis buffer???
It sounds very interesting what you say....but itīs not clear to me why I should see keratins with a cxcl12 antibody
Can you explain this any further.....Iīm really curious....
THANKS
i can't locate the reference* for the treatment at the moment but it was to include sodium metabisulfite in the sds sample buffer (i don't remember if it was with or instead of mercaptoethanol). it didn't work very well.
denise ochs published on the determination of the artifact to be keratins (can't find the reference right now*).
the binding of the antibody appears to be non-specific and/or binding of the secondary antibody.
we eliminated the reducing agent from the sds sample buffer (we maintain our proteins in buffers that contain 2mM dtt so i think you should not have to alter your lysis buffer unless it is your sds sample buffer).
* all of my old references are boxed up and a pita to look through.
this is the ochs paper: protein contaminants of sodium dodecyl sulfate-polyacrylamide gels
mdfenko on Mar 3 2010, 10:08 AM said:
denise ochs published on the determination of the artifact to be keratins (can't find the reference right now*).
the binding of the antibody appears to be non-specific and/or binding of the secondary antibody.
we eliminated the reducing agent from the sds sample buffer (we maintain our proteins in buffers that contain 2mM dtt so i think you should not have to alter your lysis buffer unless it is your sds sample buffer).
* all of my old references are boxed up and a pita to look through.
this is the ochs paper: protein contaminants of sodium dodecyl sulfate-polyacrylamide gels
THANKS A LOT
I will have a look and a try .
I will let you know
.
Hi, I am going to test this SDF-1. I am worry about this western blot because it is a pretty small protein. lymphomaW on Tue Jun 2 10:33:17 2009 said: NeutrinoQ on Mon Feb 22 23:37:47 2010 said:
Could you tell me how did you guys run the gel and transferred it (the conditions)? What gel did you use? how long did you transfer and what voltage used? Did you use NC membrane? Is the pore size 0.2um?
I found that the antibody you used (
Thank you very much!
I did a western blot of lymphoma cell lysate using anti-SDF-1(CXCL12) antibody to check SDF-1 expression. I didn't see any band attribute to SDF-1 size (10kd), however i saw a nice strong band around 50Kd. The antibody used was from R&D systems (CAT MAB350). Has anybody got similar problem when running SDF-1 western? Can anyone explain? Please help. Thanks.
Hi there,
I have gotten a similar result with brain lysate using the SDF-1 antibody from R&D (MAB350). I get no band at 8-10kDa but I get a very strong band just about/above 50 kDa. Where you able to figure out what this high molecular weight band is due to? (e.g. is it some type of oligopeptide or sdf-1 bound to something?) Many thanks for your help on this!
Best,
-NeutrinoQ