I'm trying to use SDS-PAGE to separate my 28kDa and 30kDa proteins. I tried 4%-20% gradient gel (and ran for 1 hour more than I usually do to let my band come down to the bottom), but it didn't work. I couldn't really see the difference between the bands of 28kDa and 30kDa. What should I do? Is there anything I can try to adjust? Thanks so much!
Try 12.5% gel with an appropriate gel length and run for longer period of time that will allow good resolution or else go for 2- D gel electrophoresis.
try running your protein samples in a 15 %(w/v) Tricine gel. It is best suited for getting a good separation among proteins having a molecular weight lesser than 30kDa.
you can follow this link from Nature protocols and do it :
All the best !
http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html
You could try UPLC to separate you proteins. If there are available antibodies, you could also try western-blot.
Try a narrow gradient range, such as 12.5 to 15 % acrylamide in a regular SDS-PAGE. You might get a better separation of your proteins. Tricine is a good option too. But if you want to use your protein after separation, go for a size exclusion chromatography or a C18 reversed phase at a HPLC or UPLC.
I think a gradient gel of 4-20% available in Biorad works fine. I have used this to even see a difference in mobility pattern of protein with a his-tag and then cleaving off the his tag...Just 6-10 amino acid difference.
Hi Kelly
I might be late on answering this but i guess you got to try the 2d gel electrophoresis since the molecular mass of the protein of your interest lie too close to each other so may be the charge difference can help. U already stated that u have tried til 20% gel concentration so doing conventional SDS PAGE might not help anymore. Also u can try is to replace the IEF step with a zymogram, but this requires working with a known enzyme and one of its established substrates.
Cheers!!
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