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 transferring proteins from nonreducing SDS-PAGE gel


  • Please log in to reply
7 replies to this topic

#1 hmohamma

hmohamma

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 24 March 2010 - 01:32 PM

Hi everybody,

I am having difficulty with transferring proteins from non reducing SDS-PAGE gel to PVDF membrane.
I stain my gels after transfer and the proteins are separated well enough but still there. Plus using Ponceaue red I don't see anything on teh memberane. My pre-stained marker is transferred perfectly. The only concern I have is the cell lysate. It has two fractions: one just a liquid form and the other with very high viscosity. I try to mix them well and take from both parts before running, then I just warm it at 90 for 5 min. I was wondering is it difficult to transfer the proteins in non reducing form. I had couple of times of good transfer but most of the time no luck! Any help is greatly appreciated.

Thank you guys in advance :wacko:

Hak

#2 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 24 March 2010 - 07:41 PM

Hi hak its a common problem and u need not worry a lot abt it... it will be miore helpful if u can give some more details like mol weight of the excpected protein... wb conditions.. buffers.. etc!!

Good luck!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#3 hmohamma

hmohamma

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 25 March 2010 - 06:24 AM

Hi Pradeep Iyer,

Thank you for your response. The proteins I am looking are from 24KDa-120Kda in the cell lysate of virus infected cells. Buffer I am using for transfer has Tris-Glycine in Meth-OH and H2O. So, what I am goiong to do is to run some of my samples in the reducing condition and see how it will transfer. For, people are doing it in the lab with no problem of transferring. These are what I will do. Plus I heard about CHAPS buffer fir transferr but I don't know what it is.

Thank you again

Hak

Hi hak its a common problem and u need not worry a lot abt it... it will be miore helpful if u can give some more details like mol weight of the excpected protein... wb conditions.. buffers.. etc!!

Good luck!!



#4 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 25 March 2010 - 08:13 PM

the most basic thing if the transfer efficiency is not proper is to add 0.01 - 0.1% SDS in the transfer buffer or if the pI value is high i.e more than 8.3 which is the transfer buffer pI u might have to try other buffers... but most often than not SDS shud work

Best luck!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#5 hmohamma

hmohamma

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 26 March 2010 - 11:51 AM

Pradeep Iyer
Thank you for the suggestion. Actually that was the subject of my discussion with my advisor today to use SDS! Thanks again.

Hak

the most basic thing if the transfer efficiency is not proper is to add 0.01 - 0.1% SDS in the transfer buffer or if the pI value is high i.e more than 8.3 which is the transfer buffer pI u might have to try other buffers... but most often than not SDS shud work

Best luck!!



#6 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 29 March 2010 - 12:07 AM

Oh tats great Hak... do tell us the results of your experimentation!!!! :P
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#7 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,738 posts
125
Excellent

Posted 29 March 2010 - 10:31 AM

i agree with pi that sds should help but you should not exceed 0.05% sds and you should have 20% methanol in the transfer buffer, as well.
talent does what it can
genius does what it must
i do what i get paid to do

#8 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 30 March 2010 - 10:06 PM

i agree with pi that sds should help but you should not exceed 0.05% sds and you should have 20% methanol in the transfer buffer, as well.


yes as a matter of fact we use 0.05% SDS with 20% methanol in the transfer buffer!!! :)
Support bacteria - They are the only culture some people have!!!
Cheers!!!




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

©1999-2013 Protocol Online, All rights reserved.