Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

problem with sds-page


  • Please log in to reply
8 replies to this topic

#1 marzieh

marzieh

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 08 May 2014 - 06:35 AM

Hi

1. Due to an unknown reason, low molecular weight  bands in my gel have low resolution.

I use fresh buffers.

Because the pH of Tris solutions is temperature-dependent, I adjust the pH of the solutions at room temperature and use them at the same temperature. 
The attached file is my gel picture.
What do you think about the reason? 
SKMBT_C450010429133203.jpg
 2. Why sometimes proteins dont create a thin zone in the stacking gel with a length of 1 centimeter?

Edited by marzieh, 08 May 2014 - 07:26 AM.


#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,523 posts
371
Excellent

Posted 08 May 2014 - 12:32 PM

What percentage gel are you running and what size are you after?



#3 marzieh

marzieh

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 08 May 2014 - 08:55 PM

Thanks for your attention. 

I use 15% resolving gel and my protein MW is about 8 KD.



#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,710 posts
123
Excellent

Posted 09 May 2014 - 03:58 AM

with laemmli sds-page, low molecular weight proteins are difficult to maintain sharp banding. just using high percentage acrylamide normally doesn't help. a gradient may improve the sharpness but we find that the best method for sharp separation of low molecular weight proteins and peptides is that of shagger and von jagow (tris-tricine sds-page). a 10% acrylamide gel should give you sharp banding of your 8 kDa protein.

 

however, sometimes lower molecular weight band sharpness is affected by aged sds. you can try preparing buffers with a fresh lot of sds and see if it improves sharpness.

 

but, i think you'll get better results in general if you switch to tris-tricine-sds.

 

ps. you should find the formulation by searching the forum.


Edited by mdfenko, 09 May 2014 - 03:59 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#5 marzieh

marzieh

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 09 May 2014 - 10:41 PM

Thank you very much.

In the cases where lower bands are not sharp, sometimes i encounter lack of concentration of the protein sample in the 1 cm stacking gel. What do you think about the reason?


Edited by marzieh, 09 May 2014 - 10:46 PM.


#6 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,710 posts
123
Excellent

Posted 12 May 2014 - 04:07 AM

In the cases where lower bands are not sharp, sometimes i encounter lack of concentration of the protein sample in the 1 cm stacking gel. What do you think about the reason?

only if your sample volume is too large to stack properly.


talent does what it can
genius does what it must
i do what i get paid to do

#7 marzieh

marzieh

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 12 May 2014 - 09:00 AM

Thanks for your helps  smile.png



#8 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,710 posts
123
Excellent

Posted 13 May 2014 - 05:02 AM

let us know what you do and how it works


talent does what it can
genius does what it must
i do what i get paid to do

#9 marzieh

marzieh

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 09 June 2014 - 10:59 PM

hi.

the problem was partially solved by using fresh sds






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.