polyacrylamide gel
Amit Kumar
Saturday, 18 April 2009 00:02 UTC
I am using 18 % SDS-PAGE gels for my 4.5 kDa amyloid protein…but every time I run a gel I see a big smear instead of discrete bands at the end of the gel….can anyone tell why this is happening…is something wrong with my ingredients.
Second I also see a band Patern around 50-60 kDa range I have check it with western blot and it is not protein …..what it is some kind of contamination…
Please Help
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Increase the concentration of TEMED and APS and also increase the time of polymerization of gel. I think the gel is not polymerized properly , try it and let me know
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thanks abhai for ur suggestion i will try that and let u knw…i have one more question have u ever worked with TEV protease because our recombinant amyloid protein contain TEV site and TEV protease is not cleaving it we tried everything….can you help me with that
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Are you sure you are not over-loading the gel?
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You should use a Tris-Tricine gel for low molecular weight proteins/peptides. These gels take a bit longer to run but hopefully it will be worth the wait. You will need to put your sample in tricine sample buffer before running it.
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How you control protein looks like in western.. may be 50 kd protein is aggregates.. can you detect you 5kd protein in western on the same gel?
I used to use 15% of 30 % acrylamide/bis-acrylamide for 12 Kd proteins.. so i guess 18% solution should be ok if it is not running off the gel.. (but I am not sure about it)..
Craig said right.. you might have to run gel longer to separate.. but it might also be over loading.. so what you can do.. is try diluting your sample.. and see you get clear band..
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thanks everyone for their suggestions I wil try aal these approaches..and let u guys know ..again thanks…….BUT I have one question regarding TEV protease…… our recombinant amyloid protein contain TEV site and TEV protease is not cleaving it we tried everything….can anyone help me with that…
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are you using the TEV protease to remove the fusion tag?
I guess one possible reason that the TEV protease did not work is because the TEV site in your recombinant fusion protein is not exposed to the protein surface when your protein fused with the fusion tag, thus TEV protease can not access to the cleave site.
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I want to run a native gel. But my polyacrylamide gel does not polymerize. I tried to increse APS and TEMED conc but still did not work. All my reagents are new. How much should the pH of acrylamide solution be? literatre says it is normally below 7, but my solution has a pH slightly higher? Can i reduce the pH using an acid and try?
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Hi all,
Thank you very much for participating in the Nature Protocols Discussion Forum!
To try to keep the discussion on topic, I suggest that responses to the TEV protease query are posted here.
Geny: to increase the visibilty of your query, you might wish to post it as a new forum topic? Don’t forget to add tags so that it will be found by search tools!
Thanks – and good luck with resolving your experimental issues!
Dot (forum moderator) -
Hi Xionghui,
My recombinant protein is without a fusion tag…but my protein has a very high aggregation propensity
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