Hi Andrew,

Please take a look at the link below, you may find helpful advice there. 

http://www.protocol-online.org/biology-forums-2/posts/20145.html

I hope the problem is not in your gel run. It may be with the sample preparation or sample dye. Please recheck the buffers what you are using is appropriate or not. Did you use any protease inhibitor, added before homogenization? Its important to centrifuge after homogenization (10000 rpm, 10-15 mins, 4 degree C). Recheck the loading sample buffer (pH and beta mercaptoethanol) If everything is correct, after gel run check the gel using CBB staining. Run the WB not more than 120V.

What I'm seeing is fat, uneven bands of the loading dye, but the prestained markers look fine. There may be something in the sample that is running at the dye front and interfering with the dye, like a lot of lipids or too much detergent. I suggest you try the Western blot anyway. It may be OK, except for near the bottom of the gel.

I shall be imaging the western today, hopefully I will see something. One of my proteins if 20KaD so could really do with definition at the bottom of the gel. If there are lipids in the samples is there anyway to remove them or are the sample no good?

The samples are historic (possibly 6 months old), could this sort of thing happen after this length of  storage? 

Thanks again for any help.

Just a follow up, samples were processed immediately after tissue was removed and homogenised in RIPA + protein inhibitors. They were spun down for 20mins after homogenisation and supernatent taken. All buffers are premade Thermo buffers (indate), I did not PH running or transfer buffers at the time but will check next time.

Here is a method for removing interfering substances from protein samples:

http://www.biotech.cornell.edu/sites/default/files/uploads/Documents/Proteomics_protocols/Protocol%2002_Protein%20precipitation.pdf

Are you overleading the smeary lanes?  I would not think the running buffers or gels themselves are the source of the problem as the standards look ok.  I am hoping you post what solved your problem once its working again.  Thank you!

Hi Andrew,

Please check the pH of your running buffer (should be ~8.3).

Thanks

Thank you for all your advice, I have loaded new gel today, on one gel loaded same samples but half the amount of protein. I have also ran a gel with new samples. Hopefully shed some light on the effect load was having on my run or if any possible contaminants were interfering with the run. Will post result. Thanks again.   

Have checked running buffer PH 8.45 did not alter.