SDS-PAGE with small proteins

Zhonglin Chai via methods%40net.bio.net (by Zhonglin.Chai At baker.edu.au)
Thu Nov 30 17:47:01 EST 2006


Do you have any suggestions/tips about the conditions for transferring small proteins from gel to membrane, including type of membrane, pore size, transfer buffer, voltage/current, temperature and time?

Thanks.

Chai

-----Original Message-----
From: methods-bounces At oat.bio.indiana.edu
[mailto:methods-bounces At oat.bio.indiana.edu]On Behalf Of Jess
Sent: Thursday, 30 November 2006 2:14 PM
To: methods At oat.bio.indiana.edu
Subject: Re: SDS-PAGE with small proteins 


  On 11/27/06, Marcin wrote:
> Hi everybody,
> I wonder if anyone knows how to separate and visualise small proteins
> (4-10 kDa) on a gel. I tried gradients, high percentage gels and
> Tris-tricine gels w/o any luck. When I stain with Coomassie or Ponceau
> (after transfer onto 0.2 u nitrocellulose) I can see nice bands up from
> 10 kDa. Below that point nothing shows up! Also, I tried blotting with
> specific abs w/o any success. I would appreciate any suggestions.

  Here are a bunch of "Any" suggestions.
   
  Try an 18% gel but prerun the gel (before you pour the stacking gel) for 1hr at 40mA. This sharpens the bands and compresses them such that a 7.5% gel will run as a 10% gel. Your 18% gel prerun should then run as a >20% gel. This should help you separate the smaller proteins and you can do this with Tris-Glycine or Tris-Tricine. After your prerun, then pour your stack and let it polymerize. Don't let it sit longer than about 1 hour. If you do, you could get pH diffusion between the stack and sep- this makes your bands fuzzy and you lose resolution.
   
  If you are just interested in tiny proteins, another suggestion I have is to reduce your sample volume as well as remove larger proteins. Spin your samples through an amicon or pall 10K centrifugal filter before you boil the samples. This will remove all proteins above 10kDa. You can also use the 1kDa filters to concentrate your protein sample. Smaller sample volume gives you better resolution and separation. 
   
  Also, we regularly achieve separation of proteins ~6kDa just using a good ol' fashioned Tris-Glycine 15% gel. We just don't run the loading dye all the way off the bottom of the gel. I suggest you get a protein marker with small reference points so you can monitor the progress of your run. NEB has a marker than has reference points as small as 2 kDa. Run your gel until you see good separation between the last few bands. Then at least you know you're running your gel right.
  http://www.neb.com/nebecomm/products/productP7702.asp
   
  Try the 18% gel first, with the prerun and don't run the dye off the bottom. See if that helps before you try anything else I suggested. 
   
  Question: How are you prepping your protein sample. Is there any dialysis step where you might be losing those proteins. Most dialysis tubing has a MWCO that's pretty big. Maybe its not your gel after all....
   
  Cheers,
  Jess
   

 
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