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
- - - - -

SDS Page Help


  • Please log in to reply
3 replies to this topic

#1 labtech

labtech

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 22 April 2009 - 06:09 PM

Hello All,

I have several samples of purified proteins that I want to run. However I do not want to denature the proteins. Is there another way to prepare the samples without boiling them? If I don't boil them, will the proteins bind to the gel? ( I am planning on staining with coomassie blue) Also, if I wanted a greater band separation, would I use a higher gel concentration (12%-15%) and run it with a lower voltage (~65-80V)? Some of my proteins will bind around the same point, so I want to be able to distinguish the exact locations. Thanks!

#2 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 22 April 2009 - 07:49 PM

I am not sure if I get what you are trying to do.

Why don't you want to denature the proteins, Its SDS-PAGE. Run native PAGE if you don't wana denature.

Gel conc. is related to the size of the protein, run a longer gel if you want more separation.

Best,
TC



Hello All,

I have several samples of purified proteins that I want to run. However I do not want to denature the proteins. Is there another way to prepare the samples without boiling them? If I don't boil them, will the proteins bind to the gel? ( I am planning on staining with coomassie blue) Also, if I wanted a greater band separation, would I use a higher gel concentration (12%-15%) and run it with a lower voltage (~65-80V)? Some of my proteins will bind around the same point, so I want to be able to distinguish the exact locations. Thanks!



#3 mastermi

mastermi

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 82 posts
0
Neutral

Posted 23 April 2009 - 06:56 AM

TC is right.
If you do not want to denature your protein you have to do Native PAGE.
If you want a better seperation of large molecules, use a longer gel, and if you want a better seperation of small proteins, use a Schägger gel.

#4 madrius1

madrius1

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 114 posts
0
Neutral

Posted 23 April 2009 - 06:59 AM

Proteins don't "bind" in the gel, they migrate to the point where the can't go further with the current they're provided and their weight. This is directly dependent of the acrylamide concentration of your gel.

And yes, running a Native PAGE won't denature your proteins. Keep in mind that native conditions could lend to the presence of oligomers. So you may not find only one sharp band, as you would in a denaturing SDS PAGE. Boiling your samples would denature them, so no boiling :rolleyes:




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

©1999-2013 Protocol Online, All rights reserved.