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
- - - - -

smear on gel after tca precipitation


  • Please log in to reply
3 replies to this topic

#1 yja97

yja97

    member

  • Active Members
  • Pip
  • 11 posts
1
Neutral

Posted 17 June 2009 - 10:08 AM

Hi. My problem is just like the title.
SDS PAGE of my protein after TCA ppt makes smear instead of discrete bands.

I precipitated my protein sample with tca + na-deoxycholate, followed by washing with acetone and resuspended with 1X SDS-PAGE loading buffer.
At first the sample is yellow, so I added a small amount of 1N NaOH until the color changed to blue.
I need my sample for western blotting, and the signal intensity is good but the shapes are still long circle. I need to make them bands.

How do you think I can solve this problem?

Thanks in advance.

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,709 posts
123
Excellent

Posted 17 June 2009 - 11:56 AM

you are probably experiencing incomplete solubilization of your protein.

the yellow indicated that you still had acid in your protein. using naoh is harsh and may cause some decomposition of the protein. we use 0.5M tris (pH9 or base, whichever you prefer) to neutralize remaining acid.

both of these can cause smearing.
talent does what it can
genius does what it must
i do what i get paid to do

#3 yja97

yja97

    member

  • Active Members
  • Pip
  • 11 posts
1
Neutral

Posted 17 June 2009 - 01:11 PM

Thank you so much.
If insolubilization matters, how can I increase the solubility?
Longer vortexing and heating would be helpful?


you are probably experiencing incomplete solubilization of your protein.

the yellow indicated that you still had acid in your protein. using naoh is harsh and may cause some decomposition of the protein. we use 0.5M tris (pH9 or base, whichever you prefer) to neutralize remaining acid.

both of these can cause smearing.



#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,709 posts
123
Excellent

Posted 18 June 2009 - 08:33 AM

Thank you so much.
If insolubilization matters, how can I increase the solubility?
Longer vortexing and heating would be helpful?


make sure you properly neutralize the tca then heat for an appropriate amount of time.

keep in mind that at least part of your problem was caused by using naoh to neutralize. naoh hydrolyzes proteins.
talent does what it can
genius does what it must
i do what i get paid to do




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

©1999-2013 Protocol Online, All rights reserved.