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zeeshan's Content

There have been 4 items by zeeshan (Search limited from 26-October 19)


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#173822 SDS page staning issue

Posted by zeeshan on 13 March 2015 - 10:10 AM in Electrophoresis

Lysis buffer component: DTT, PMSF, MgCl2, KCl, HEPES, nonyl phenoxypolyethoxylethanol (NP40). sample buffer component: Tris, SDS, Glycerol, Bromophenol Blue and betamercaptoethanol. I am using tissue homogenate.  




#173793 SDS page staning issue

Posted by zeeshan on 12 March 2015 - 02:54 AM in Electrophoresis

I heated my protein at 70c for 10min  in presence of sample buffer and ran gel. I got the same results as earlier (no bands in the bottom of gel). Please advise. 

Attached Thumbnails

  • new gel image1.jpg



#173763 SDS page staning issue

Posted by zeeshan on 10 March 2015 - 11:33 PM in Electrophoresis

Bob, no its not a sticky agglomerate. I changed the protocol this time and now I am heating at 37 in presence of sample buffer for 30min. BUt I am facing a new issue. I ran gel this time and got bands but the bottom of the gels are quite blank. I proceeded with western blot I got expression but at wrong place. Why is this so?

 

mdfenko, yes earlier I boiled without buffer. but I dont think the sample are degraded. What I did was i made a shot spin of that sample and collected the supernatant and ran a gel I got bands. If it would have been degraded why would I get the bands. But yes next time I will heat at 70c. However I have a new issue. I have changed my protocol and now I heat at 37c for 3o min in presence of sample buffer. after the gel I observed that bottom of gel is quite balnk with faint bands. I did western blot also but I got expression but at wrong place. Why is this so?     




#173540 SDS page staning issue

Posted by zeeshan on 25 February 2015 - 11:32 AM in Electrophoresis

I am doing SDS page for my sample. I use dignam buffer for lysis. Protein quantification shows spec reading (something around 1.125) after that I heated my sample at 95c for 5 min. I am loading 15micro/g per well. but to my utter realisation I am not getting a single band after staning. I ntocied one thing after sample kind of agglomerate i can see in my sample. Can someone tell me what wrong I am doing. I did all pH checking and evrthing. I dont know what is the problem?





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