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
- - - - -

Large proteins in Western


  • Please log in to reply
12 replies to this topic

#1 criscastells

criscastells

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
1
Neutral

Posted 14 September 2012 - 08:58 AM

I want to analyze some large proteins using WB and I have read different opinions about how to do it.
On one hand I have found that it is improtant to decreasde MeOH concentration to 10% and add SDS in transfer buffer up to 0.1%. On the other hand there are some bibliography that indicate not to change transfer buffer.
Additionally I have also read that I may change voltage and time of transference.

I am confused. Can someone give me some advice?

Thanks you very much in advance

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,817 posts
137
Excellent

Posted 14 September 2012 - 11:24 AM

depends on how large the protein is.

you can reduce the acrylamide concentration of the gel

add up to 0.05% sds and 20% methanol to the transfer buffer

transfer for more time (how much more should be determined by trial)

remember that you may never get 100% transfer

here is a useful book:

Attached Files


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

#3 science noob

science noob

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 281 posts
20
Excellent

Posted 15 September 2012 - 12:54 AM

What's the size of your protein of interest?

I was optimising conditions for the transfer of a ~400kDa protein and had some decent transfer with the following conditions:

5-6% resolving gel (homemade polyacrylamide) - run the 250kDa ladder to halfway down the gel during SDS-PAGE (it's advisable to keep a housekeeping protein on the gel - I used tubulin)

2x Tris-glycine transfer buffer - double the Tris and glycine content. I kept the methanol to 20% (will most likely try to decrease this to 10% as suggested on this forum) and didn't add SDS (might try to add it in the buffer). These 2 additions might make it even favourable.

Wet transferred it overnight 30V followed by another 4 h transfer at 100V. Its important to use a cold room or at least an ice bath because the buffer could heat up (especially the 100V transfer). You can change the ice block to a new one to maintain low temperatures.

Hope this helps! Posted Image

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,817 posts
137
Excellent

Posted 17 September 2012 - 05:13 AM

if you add sds to the transfer buffer then you need more methanol to properly strip the sds from the protein after it has done its job in facilitating transfer. it would be best to allow the transfer buffer to remain at 20% methanol.
talent does what it can
genius does what it must
i do what i get paid to do

#5 criscastells

criscastells

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
1
Neutral

Posted 19 September 2012 - 06:30 AM

Thanks to very much for all your advice. My proteins are not as large (200KDa) so I think that I do not need an over night transfer. I will check it but I will add SDS to the normal transfer.

Thanks again! (hope work ;P)

#6 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 19 September 2012 - 01:51 PM

200 kDa is nothing. Don't worry. You can use normal transfer conditions with increasing a bit the transfer time. For 400 kDa or 600 kDa (tyroglobulin) then we are talking about changing conditions.

#7 science noob

science noob

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 281 posts
20
Excellent

Posted 19 September 2012 - 06:02 PM

Yup agreed with @ascacioc. 200kDa is considered as a fairly transferable size as most ladders have 250kDa as the largest marker. Transfer under normal conditions (10% SDS-PAGE gel transfer for 1h at 100V in the cold room/ice bath...at the end of the transfer, if your 250kDa has fully transferred from gel to membrane, then you know it's definitely transferred) and let us know how it turned out Posted Image

#8 criscastells

criscastells

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
1
Neutral

Posted 20 September 2012 - 07:12 AM

I wanted to change transfer conditions because after the transference I can see big proteins still in the gel (coomassie staining).
I have tried changing tranfer buffer (10% MeOH and 0,1%SDS) and there are no proteins in the gel (GREAT!!). But now my problem is that the staining of marker proteins have dissapeared so I do not know if the band corresponds to 200KDa (WEIRD!).

I'm suposed to buy another marker with chemiluminiscence.

Thanks you very much for all your help!

#9 science noob

science noob

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 281 posts
20
Excellent

Posted 21 September 2012 - 02:04 AM

I wanted to change transfer conditions because after the transference I can see big proteins still in the gel (coomassie staining).
I have tried changing tranfer buffer (10% MeOH and 0,1%SDS) and there are no proteins in the gel (GREAT!!). But now my problem is that the staining of marker proteins have dissapeared so I do not know if the band corresponds to 200KDa (WEIRD!).

I'm suposed to buy another marker with chemiluminiscence.

Thanks you very much for all your help!


You won't be able to transfer all protein fragments from gel to membrane in the real world. You don't need all proteins to be transferred to get decent/good staining.
The marker proteins you're referring to = standard protein marker??

#10 criscastells

criscastells

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
1
Neutral

Posted 01 October 2012 - 02:09 AM

Yes, the standard protein (all of them) are lost during the transference.

#11 science noob

science noob

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 281 posts
20
Excellent

Posted 01 October 2012 - 05:06 AM

Yes, the standard protein (all of them) are lost during the transference.


Lost from the gel or membrane/blot?

#12 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,817 posts
137
Excellent

Posted 01 October 2012 - 06:05 AM

what is the membrane type, pore size?

did you back it with a second membrane?

do you see anything on the membrane? have you stained with ponceau?

sds, if not stripped from the protein during transfer, will interfere with binding. that's why the manual recommends not exceeding 0.05% and adding up to 20% methanol in the transfer buffer.
talent does what it can
genius does what it must
i do what i get paid to do

#13 criscastells

criscastells

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
1
Neutral

Posted 01 November 2012 - 02:41 AM

The standards do not appear in the membrane neither in the gel. Probably I use too much SDS. I know it is not possible to transfer completely all the proteins but my problem is that the big ones (from approximatelly 100KDa) remain in the gel using normal conditions.

I will try again using 20%MeOH ans 0,01%SDS. I think I will also increase transfer time.




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

©1999-2013 Protocol Online, All rights reserved.