Hi, i have run many SDS-PAGES during working on my projects but recently I faced a problem.to check the purity of a recombinant peptide I am loading that after purification with Ni-NTA but after staing the gel all bands are duiffused and in so strange shape. There are no sharp bands and just bands like branches!I tried to prepare all buffers freshly and also I run at 80V for the first layer and 120 for the second but stil I couldnot overcome. could you please help me in this case?
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#2
casandra
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Posted 30 June 2011 - 08:48 AM
Hi pEXP23....welcome to bioforum....it would help if you can post an image of your stained gel as well as more details about your protocol....meanwhile, don't despair...Hi, i have run many SDS-PAGES during working on my projects but recently I faced a problem.to check the purity of a recombinant peptide I am loading that after purification with Ni-NTA but after staing the gel all bands are duiffused and in so strange shape. There are no sharp bands and just bands like branches!I tried to prepare all buffers freshly and also I run at 80V for the first layer and 120 for the second but stil I couldnot overcome. could you please help me in this case?
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#3
pEXP23
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Posted 01 July 2011 - 10:29 PM
Thank you so much for your kind welcome:) Please find my gel image. I should mention that i had stored my samples at 8 degree for about 4 days. and after that I found out that it has some presipitate. Before this the gel has two sharp bands.
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#4
bob1
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Posted 02 July 2011 - 02:35 PM
HOw big is your peptide, and what percent gel did you run?
Protein samples should never be stored at fridge temperatures unless they are in a lyophilised state. Always store at a minimum of -20 deg C.
Protein samples should never be stored at fridge temperatures unless they are in a lyophilised state. Always store at a minimum of -20 deg C.
#5
pEXP23
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Posted 02 July 2011 - 10:32 PM
The peptide is about 13.5KD and the percentage of gel is 17%.So maybe the protein is degraded
#6
Adrian K
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Posted 03 July 2011 - 07:22 AM
IMHO, it looks like not well polymerized GEL
I would suggest:
Change to newly prepared /purchased APS. Use it fresh.
Use a multichannel pipette to load your acrylamide in between the glass when you make your gel. DO not load in one-side.
Use a lower voltage to avoid overheating, better you run your PAGE inside ice-box (I always do that).
I would suggest:
Change to newly prepared /purchased APS. Use it fresh.
Use a multichannel pipette to load your acrylamide in between the glass when you make your gel. DO not load in one-side.
Use a lower voltage to avoid overheating, better you run your PAGE inside ice-box (I always do that).
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"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#7
pEXP23
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Posted 03 July 2011 - 10:26 AM
Thank you for your suggestions. But in fact I did all of these using fresh buffers and APS. And also one time I put ice outside the tank and also reduced the voltage to 100v:( anyway I will try it again.
#8
Melvin J Noe Gonzalez
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Posted 19 August 2011 - 11:55 AM
I dont know if it still going to help but I had a similar problem, and the reason was that the ph of the samples I was loading was too acidic, so after neutralizing it a little bit more, everything was perfect
Edited by Melvin J Noe Gonzalez, 19 August 2011 - 11:58 AM.
