Gel Shift Assay--Probe in Wells
genecutl at mendel.berkeley.edu
Mon Aug 8 23:24:05 EST 1994
In article <325p0v$h5p at usenet.INS.CWRU.Edu>, pxb26 at po.CWRU.Edu (Peter
> What are the some common causes for probe not leaving the wells in gel shift
> What are some suggestions for avoiding this problem?
Probe gets stuck in the wells for two reasons that I know of:
1) Sometimes probe is found in the wells independent of protein being
added, i.e. it can occasionally be seen in lanes in which the probe is
being run alone. I think this is probably due to funky polymerization
of acrylamide in the wells. I always wash out the wells with buffer
immediately after removing the comb to get rid of unpolymerized acrylamide
which may come back to haunt me. It seems to work.
2) Probe can be retained in the wells in a protein-dependent manner.
With almost any protein, if you add enough of it, it will trap the DNA
in the wells. This is generally non-specific. Most of your standard
protein-DNA complexes are small enough to enter the gel. If you add a
huge amount of protein, you'll get non-specific protein-protein and
protein-DNA binding that leads to the formation of a large aggregate.
This can also happen with lesser amounts of protein if your protein
is somewhat shitty (to use a technical term) and has a tendency to
precipitate. To help get rid of non-specific aggregation/precipitation
I always inculde 0.1% NP-40 in my gel and running buffer. Also, you
can reduce the protein concentration and/or find better quality protein.
...while making feet for children's shoes
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