Dear everyone,
I would like to separate WB proteins within the range of 80-265 kDa using directly a unique polyAcrylamide gel, maybe in a 6 % gel. Do you think this is possible?
Many thanks in advance.
You should consider using a 7-8% gel. I think 6% gel is too weak.
I have done it with 220 kDa on 10% too, by slightly over running the gel (proteins) fro atleast 10 mins. Although 8% would be the ideal gel but it agains depends on the basal level conc. of that protein, if less then your band would come diffused in 8%. Same thing happend to me and that the reason i had to adapt to 10% gels
Hi Alberto,
I don't know if it is too late, but I've separated proteins of those weight perfectly in 7,5% gels. Good luck!
I have separated considerable number of proteins in that range using 8% polyacrylamide gels. They work fine. In case of high molecular wt. protein you should consider running the gel for extra 20-30 mins.
I'm planning to use the pre-cast gels by Bio-Rad (4-15%) and then we'll see which results we'll get.
Many thanks again to everyone.
Oh dear, thats not a problem. I made western blot for DNA-PK that is 460 kDa. I prepared 5% SDS-PAGE Gell. And left it for 150 min (130V). We also use GenScript eBlot Protein Transfer (for this gel I set it for 20 min transfer, typicaly for proteins at size of 60 kDa it takes 7 min). So your problem may be A. Gel density, or B. transfer time.
I feel the best would be to use gradient gels. You can simply buy them from Biorad and the range you are looking should be able to separate in 5-10% gels. Hope it works. Good luck
Many thanks for the answers.
Actually I am using pre-cast gradient gels and they work very nice.
Is your pre-cast gradient gel not separating properly the protein of your interest? I also use pre-cast gel (8-16% Tris-Glycine) from Biorad which has a range of 10-250 kDA but it goes beyond that. You can try doing this. Also you can stain the gel with Coomassie Blue to make sure you found out the band for that particular protein. Good luck.
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