Smear in Native PAGE - (Apr/16/2006 )
HI, I use 10% Native PAGE to run my TRAP assay product. THe product contains DNA fragments of sizes 42bp, 48, 54..+6bp...so on. I anticipate to see a ladder pattern in the positive control which shows telomerase activity which I used to but untill recently when suddenly the laddder disappered and I see just a smear in the positive control lane!! I tried using new 10%APS and tried 12.5% gel. But no improvement. Any sugegestions?? This problem is driving me nuts!
it appears that the bulk of your material is at the top of the gel. if this was a protein gel then the answer would be obvious, the protein is aggregated. since this is nucleic acid it would appear that your sample is precipitated (for whatever reason). rather than using a higher percentage gel, you should use a lower percentage of acrylamide to resolve higher mw sample. but first you may want to find a way to resolubilize your sample (reprecipitate with etoh and acetate or heat sample or...?).
sorry for rambling on but hopefully some of this will help.