Gel smear in kinase assay

Glenn Jenkins gjenkins at facstaff.wisc.edu
Tue Oct 29 13:09:12 EST 1996


In article <chall.846520511 at blue.weeg.uiowa.edu>,
chall at blue.weeg.uiowa.edu (C. Hall) wrote:

:         I just ran an in vitro kinase assay that produced perplexing 
: results.  I immunoprecipitated my kinase from monocyte-derived 
: macrophages, gave it enolase as a substrate and hot ATP-gamma, and then 
: ran the results (which should be mostly labeled enolase) on a minigel.  I 
: ran the free nucleotide off of the end of gel and then made an autorad.  
: There were no distinct bands, but each lane showed an equal distribution 
: of label, ie., the entire lane was one big band.  Anyone have any clue 
: what happened?

A couple of things: 

1) Did you stain the gel?
   if so what did the proteins and markers look like? Was enolase present?

2) How did you treat the gel after you ran it? I usually stain, then
destain extensively. This get rid of background which may account for your
results.

3) How much enolase did you use? If you loaded to much, the protein will
spread over into the next lane.

Hope this helps.


Glenn Jenkins
University of Wisconsin



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