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Western problem - very challenging - distorted band, non-reproducible, generally awful (Dec/02/2005 )

Hi guys,

I have a big problem at the moment (or it feels big in my own little WB world). I have been looking at a 75kDa protein for some years now, which appears to be insoluble in skeletal muscle. Therefore, when we do protein extractions from skeletal muscle biopsies, we homogenise the tissue, spin it down to get the S/N and then resuspend the pellet in 2xSDS sample buffer with 8M urea and 2M thiourea. I then load ~35ug of the pellet on SDS-PAGE gels and blot to PVDF and probe with a polyclonal antibody, then develop with ECL+.

This has been working ok for the most part, but with some new samples that i have extracted, it is not looking good. I have attached one of my blots for everyone to see. The band i am interested in is the one above the big massive band (which may be a problem in itself) indicated by the arrows. The biggest problem is the variability in the band intensity and shape. I know that the variation is not real, and does not follow any pattern because i have repeated this many times and have never got the same result (but its always been as bad). The band is often distorted and results occasssionally in half a band. The worst examples are highlighted in red boxes. The protein i am working on is largely unknown with no published protein work to date, and appears to me to be extremely difficult to work with. There are eight known isoforms expressed in skeletal muscle so some of the smaller bands may well be real and not non-specific.

I have tried almost everything that first springs to mind: diluted sample, less sample, extra BME/DTT, nitrocellulose (which is much worse interestingly), PVDF, transfer buffer pH (the PI of my protein is 8), wash steps etc.

The odd thing is, when i run a few samples on a mini-gel, it all looks ok...ish, hence why i scaled up to include all my samples. An additional point to note is that these samples are extracted using a standard phosphatase inhibitor buffer where as my previous samples were not, which could be causing problems but i think its unlikely. It also seems possible that the urea/thiourea is causing problems but i dont honestly think so - i have tried heating the samples to different temperatures prior to loading and found no major differences.

Any ideas people????? Our latest thinking is that its a gel problem - possibly something to do with gel temperature, or that the sample needs to be cleaned up.

Please, please, please give me your thoughts.

-Dukey-

Hi!
I once had this problem and it was the protein itself. Maybe you will need to investigate the pI of your protein and the hydrophobicity of the particular band.

Also, have you tried adding a little tricine to your buffer? Maybe changing the pH just a little bit?

It really looks as though the protein is sticking to the side of your wells!

Good luck - hope any of this helps!

Cindy

-biokmst-

Hi,

Thanks for your reply, i have thought about it being a hydrophobicity issue. I have tried a CAPSO buffer (pH10) which didnt seem to help much at all. I agree that it is probably a problem with the actual protein and the way it behaves. Did you manage to sort your issue out?

Luke

-Dukey-

QUOTE (Dukey @ Dec 7 2005, 02:21 PM)
Hi,

Thanks for your reply, i have thought about it being a hydrophobicity issue. I have tried a CAPSO buffer (pH10) which didnt seem to help much at all. I agree that it is probably a problem with the actual protein and the way it behaves. Did you manage to sort your issue out?

Luke

Hi!
Thanks for asking....I did - by dropping the his-tag. I believe now that the his-tag was interferring with one of my zinc binding sites by way of a histidine. Now that the tag is off, the protein expresses in all cell lines beautifully! But now, I have to use my imagination to purify the protein!

PS...do you go to Duke? I did a post doc at Duke in Pathology!


Cindy

-biokmst-

haha no i dont go to Duke, i am at the University of Nottingham, UK.

-Dukey-

If your protein looked worse on nitrocellulose, it might be because nitrocellulose binds less protein than PVDF, so I am thinking it might help to try and bind as much protein as possible? I know that's not the root cause of your problem, but it could help in the meantime. You could add more MeOH to your transfer buffer to increase binding and make sure there is no SDS in it. You could also transfer longer.

-WAstate-