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?
Submit your paper to J Biol Methods today!
7 replies to this topic
#2
casandra
-
- Global Moderators
-
- 5,034 posts
carpe diem by the jugulum
56
Excellent
Excellent
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?
...
"Oh what a beauteousness!"
- hobglobin, personal comment about my beauteous photo......
- hobglobin, personal comment about my beauteous photo......
#3
pEXP23
-
- Active Members
-
- 8 posts
member
-1
Neutral
Neutral
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.
Attached Files
-
1.bmp 940.81KB
196 downloads
#4
bob1
-
- Global Moderators
-
- 5,516 posts
Thelymitra pulchella
371
Excellent
Excellent
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
-
- Active Members
-
- 8 posts
member
-1
Neutral
Neutral
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
-
- Global Moderators
-
- 707 posts
Legendary Graduate Beggar
28
Excellent
Excellent
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).
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."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
..."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
-
- Active Members
-
- 8 posts
member
-1
Neutral
Neutral
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
-
- Members
-
- 1 posts
member
0
Neutral
Neutral
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.
