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
- - - - -

manual 2D electrophoresis


  • Please log in to reply
8 replies to this topic

#1 biochemscientist

biochemscientist

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 26 November 2013 - 09:19 PM

Hi folks,

I am doing 2D electrophoresis but not with IPG strip this time but simple PAGE run before 2D, strip is cut from PAGE gel and placed horizontally on second dimension gel.

Normally the articles that i read used smaller spacer width in the second dimension i.e first dimension run in 1mm spacer plate and second dimension run in 0.75mm spacer plate. But i do it oppositely i.e my first dimension gel is run in 0.75mm spacer plate and second dimension in 1mm spacer plate. I do it because my first dimension gel strip easily slides down the 1mm spacer plate in second dimension. I polymerize the second dimension gel first and then slide the first dimension strip onto it.

Now i have noticed oen thing that my two gels are not perfectly i contact with each other even though i overlay the gel and strip with stacking gel and then 0.5% agarose. and i think because of this i am getting a lot of horizontal streaking.

Can anyone please guide me regarding this. and if you have any video of such manual 2D methods please share it with me

 

Thanks



#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,788 posts
132
Excellent

Posted 27 November 2013 - 05:50 AM

rather than overlaying with stacking gel you should insert the gel strip through the not yet polymerized stacking gel to ensure that there are no gaps.

 

if you are afraid that polymerization may affect your proteins then you can pour a layer of stacking gel (overlay to ensure a flat surface), allow it to polymerize, then insert you gel strip and push it against the softer stacking gel to ensure complete contact.


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

#3 biochemscientist

biochemscientist

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 27 November 2013 - 07:22 PM

ok i will try like this.

Thanks alot !



#4 biochemscientist

biochemscientist

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 07 December 2013 - 12:34 PM

Hey friends,

 

this is the result of my 2D SDS PAGE please suggest how to improve this.

 

Regards,



#5 biochemscientist

biochemscientist

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 07 December 2013 - 12:36 PM

Hey friends,

 

this is the result of my 2D SDS PAGE please suggest how to improve this.

 

Regards,

 

Attached Thumbnails

  • Untitled.png
  • Untitled.png


#6 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,475 posts
251
Excellent

Posted 07 December 2013 - 01:22 PM

I don't know what you are trying to achieve by this. With standard 2-D gele, the first separation is by pI and the second by molecular weight. It seems as if your technique will (at best) separate by molecular weight twice.



#7 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,788 posts
132
Excellent

Posted 07 December 2013 - 03:31 PM

are you running native page in the 1st dimension (that's what i was assuming)?

 

it appears that you have aggregates in your initial sample. if so, they are solubilizing during the run, causing smearing which will cause very broad bands in the 2nd dimension.

 

you should ensure solubilization is as complete as possible then clear the sample prior to application to the 1st dimension.


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

#8 biochemscientist

biochemscientist

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 07 December 2013 - 08:58 PM

Yes the first dimension is a native page in which i am separating the complexes and in second dimension i am resolving those complexes. How can i clear my sample before loading as i have tried to solubilize the maximum amount of my proteins



#9 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,788 posts
132
Excellent

Posted 09 December 2013 - 04:20 AM

you can clarify your sample by centrifugation or by filtration. this should remove any undissolved or particulate material.


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.