From: mangalam@SALK-SC2.SDSC.EDU (Harry Mangalam)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Mobility Shift Assay
Date: 18 Dec 92 18:46:33 GMT
Distribution: bionet

>LCNAVOT@WEIZMANN.WEIZMANN.AC.IL (Nir Navot) writes:
>> I've been trying to perfect the conditions for running a Mobility Shift 
>Assay.

And Jorge L. Sepulveda replies:

>Here are a few tricks,
>
>1. I always purify my ds oligos, either after annealing or after 
>filling-in with Klenow; if you choose the hot nucleotide as the last to be 
>filled in, you know that all you labeled probe is double stranded and 
>blunt ended (although the incorporations are lower, but I use only 10,000 
>to 20,000 cpm/lane)
>2. Maybe for the above reason, I find filled in probes to have much less 
>background than end-labeled
>3. For some probes, 1 5g/reaction of E. coli DNA is better, for others I 
>use 1 5g/5l of dIdC
>4. I use 1/4 TBE and run it at 10W, 4!C
>5. I just add dyes (XC+BB) to the sample immediatly before loading, 
>because there is enough glycerol to sink the sample
>6. Be carefull with the order of addition of reagents; I do buffers, 
>competitor dna, nuclear extracts, wait 5-10', add hot probe
>7. The amount of glycerol (may be because it interacts with borate) can 
>cause a lot of smearing and background; keep it less than 4% in the 
>reaction


My $ 0.02 are:

   It all depends on the protein that you're working with!  While in most
cases, I agree with Jorge, my experience has been:  

1)  You can use 1/2X TBE, 1/4XTBE, or even less (or if your protein is
refractive to lowering the concentration of TBE, you can also use other
buffers).  A friend who was trying to get glucocorticoid receptor to bind
found that it would only bind in a Glycine-based buffer.

2)  In another situation, the addition of the loading buffer, specifically
the Xylene Cyanol, completely disrupted the binding.  Try other dyes or
bite the bullet and load w/o dyes (a pain and a half, but ya gotta do what
ya gotta do).

3)  Competitor DNA makes a _HUGE_ difference, in specificity, in position
of band shift, in tightness of the band, etc.  Unless there's a very good
reason not to, I used sonicated salmon sperm or calf thymus DNA.  It's
cheaper than dI/dC and it provides the heterogeneity to assure that you are
detecting specific binding.  

4)  Relating to 3), you ABSOLUTELY MUST do concentration curves of
competitor DNA to determine how stable the shifted bands are.  I've seen
supposedly specific bands do the most beautiful sigmoid curves across a gel
of increasing [competitor DNA]. A crude depiction is shown below for the
same reaction differing only in amount of competitor DNA.

            Competitor DNA (ng)
.01  .03  .1   .3    1    3    10   30  100  300 
-------------------------------------------------

===  ===                           +++  +++  +++
          ===       ---  ---  ---  ---  ---  ---     
               ---            +++
          ---  ===       +++  
+++  +++  +++  +++  +++     
                    ===
---  ---                 ===
                              ===  ===  ===  ===


This kind of result can indicate a range of things from competition for
sites, to multiple binding sites to association with other proteins to
??????.

   In the course of my long and hateful relationship with gel shifts, I've
been bitten by a variety of gotchas, so many that I consider gelshifts to
be among the least useful (and most used) techniques in molbio.  Unless you
have a  well-defined system where you absolutely know what the constituents
are, assuming anything only on the basis of a gelshift is asking for your
(paper, post-doc, thesis) to be delayed considerably or put in a
retractable state.  As such, I would use gelshifts only in desperation or
as a very early step in trying to find 'anything'.  

   But despite the above, the world (and gel shifts) will continue.

Cheers,
Harry

Harry Mangalam                                   Vox:(619) 453-4100, x250
Dept of Biocomputing                                   Fax:(619) 552-1546
The Salk Institute                        1'   mangalam@salk-sc2.sdsc.edu
10010 N Torrey Pines Rd                   2'        hjm@salk-sgi.sdsc.edu
La Jolla CA 92037                         3'         mangalam@salk.bitnet


Newsgroups: bionet.molbio.methds-reagnts