13 replies to this topic

[lymphomaW]

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.

[Carlton H]

CXCL12's receptor, CXCR4, is right around 46kd...  Are you possibly seeing the receptor-bound protein?  The receptor + protein would be in the ballpark of 50kd.

-Carlton

[lymphomaW]

[quote name='Carlton H' date='Jun 5 2009, 12:23 PM' post='26070']
CXCL12's receptor, CXCR4, is right around 46kd...  Are you possibly seeing the receptor-bound protein?  The receptor + protein would be in the ballpark of 50kd.

Thanks for your reply. You could be right. Would it also be possible the multimers of SDF-1? I'm re-running western using 150mM DTT in my loading buffer instead of 50mM to see whether the problem still there. Do you think will this solve the problem? Any advice?

lymphomaw

[Carlton H]

I'm not going to pretend to be a structural biologist, but more DTT should chemically break disulfide bonds.  I'm not familiar with the tertiary structure of SDF-1, but if it forms oligopeptide interactions based on disulfide bonds (which would be reasonable), then increasing the concentration of DTT may give you better results.

...did you try it?  How did it turn out?

Cheers,
-Carlton

[lymphomaW]

Carlton H, on Jun 11 2009, 08:49 PM, said:

I'm not going to pretend to be a structural biologist, but more DTT should chemically break disulfide bonds.  I'm not familiar with the tertiary structure of SDF-1, but if it forms oligopeptide interactions based on disulfide bonds (which would be reasonable), then increasing the concentration of DTT may give you better results.

...did you try it?  How did it turn out?

Cheers,
-Carlton

I tried it. It didn't make any difference. I guess if SDF-1 indeed forms oligopeptide, it may not formed via disulfide bond. could it be hydrophobic interaction? any idea to break the interaction?
Thanks.

[NeutrinoQ]

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

[bluedoozer]

This is really strange, I used the same antibody and got the same f*** 50kDa band.
An interview with R&D was inconclusive, they even don´t know.

Is there anybody out there who can help us???

Personally I don´t think what we observe is a receptor ligand form. From my understanding there is no strong enough connection between receptor and ligand, surviving 95°C boiling and DTT/ß-ME and SDS....

So please...anyone who can help??

[laurequillo]

I dont think you can see the interaction of the receptor after the boiling in SDS with DTT.
Could it be an inespecific band?? Do you have any way to be sure about the specificity of your 50kDa band?

[bluedoozer]

laurequillo, on Feb 23 2010, 10:34 PM, said:

I dont think you can see the interaction of the receptor after the boiling in SDS with DTT.
Could it be an inespecific band?? Do you have any way to be sure about the specificity of your 50kDa band?

That the point
i don´t think it´s specific, but pos. controls like heart kidney lung  and liver show us the band
as well as our test samples

so it´s strange that our pos. controls show only at 50k this band

[mdfenko]

the 50k band is most likely an artifact that shows up more and more as the reducing agent ages. it has been identified as, most likely, keratins from dust.

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).

[bluedoozer]

mdfenko, on Feb 24 2010, 09:24 AM, said:

the 50k band is most likely an artifact that shows up more and more as the reducing agent ages. it has been identified as, most likely, keratins from dust.

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 don´t add to your lysis buffer???
It sounds very interesting what you say....but it´s not clear to me why I should see keratins with a cxcl12 antibody
Can you explain this any further.....I´m really curious....
THANKS

[mdfenko]

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

Edited by mdfenko, 03 March 2010 - 09:10 AM.

[bluedoozer]

mdfenko, on Mar 3 2010, 10:08 AM, said:

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

THANKS A LOT
I will have a look and a try .
I will let you know
.

[alex2815]

Hi, I am going to test this SDF-1. I am worry about this western blot because it is a pretty small protein.

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 (R&D systems MAB350 http://www.rndsystem.../pdf/mab350.pdf) is not for western blot, it might cause the problem.

Thank you very much!

lymphomaW, on 02 June 2009 - 02:33 AM, said:

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.

NeutrinoQ, on 22 February 2010 - 03:37 PM, said:

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