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 protein

ATM large protein SDS-PAGE

  • Please log in to reply
3 replies to this topic

#1 Guy

Guy

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 23 October 2011 - 04:23 AM

Hi There,

I'm trying to detect ATM (460 Kd) from Arabidopsis. I think I managed the first time but the WB signal was really high in the membrane. When I try to run it longer, I get all lower bands (I use HiMark ladder, Invitrigen) smearish in the gel. I ran it for about 5 h in the cold room but I'm not sure if it's wise because of the precipitation of the SDS, and still have the lower bands smeared like hell. The question is, can I run it O.N in 4oC in low current. Is there any other advise? (we use Tris-HCl biorad, 4%stacker;6%resolving).
Thanx a lot,
Guy

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,728 posts
399
Excellent

Posted 24 October 2011 - 03:49 PM

Presuming you actually mean tris-glycine (rather than tris-HCl), have you tried other gel types such as tris-tricine? I would not use a stacker, you probably want as much of the gel as you possibly can get, to allow decent resolution. Try running a longer gel.

With a 6% gel, you won't get any resolution of bands below about 70 kDa, and if you aren't interested in them, don't worry about them smearing/not resolving.

460 kDa is huge, you will probably struggle to get good gels of it, even 6% is probably too high a percentage.

You should be able to run it in the cold room with no problems, the SDS shouldn't precipitate while running, as the current will generate heat. You will need to have SDS and methanol in the transfer buffer, and you would be well advised to try different membrane types.

#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,785 posts
132
Excellent

Posted 25 October 2011 - 09:59 AM

if you want to ensure that the detergent doesn't crystallize in the cold then you can use lithium dodecyl sulfate instead of sds. it's a little more expensive but it's soluble in the cold.

you may also want to reduce your acrylamide to 5% (you could also reduce the percentage of crosslinker).

if you want to sharpen the lower molecular weight smear then you could run a gradient gel.
talent does what it can
genius does what it must
i do what i get paid to do

#4 Guy

Guy

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 26 October 2011 - 11:06 AM

Hi guys,

Thanx a lot, we'll start with the pre-cast Tris-acetate 3-8% Invitrogen gels and see how it goes.

Cheers,
Guy





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

©1999-2013 Protocol Online, All rights reserved.