About His-tag purification

arnaud.garcon at syngenta.com arnaud.garcon at syngenta.com
Thu Apr 13 02:49:21 EST 2006


 
Hi,
> 
>  I'm expressing a HIS-tagged 16KDa protein in E.coli
> 
> I got the target protein using the SDS-PAGE analysis

How did you stain it? Coomassie? Silver stain?  Anti-His Blot?

> 
> However, the UV absorbance is 260 nm but not 280

Are you saying the lambda max is 260 or you have *no* absorbance at 280?
If the later I would check your spectrophotometer.
If the former you may have some contamination with nucleotides. Is your
protein likely to bind to DNA/RNA?
Have you done a bradford or BCA titration to check whether concentration
calculated from OD 280 corresponded to that calculated with different
method?

> 
> I wander wether the protein binding with DNA/RNA in E.coli lysate
> 
That may not work but how about staining your SDS gel with ethidium
bromide if you're convinced you have contaminating DNA?


A.-



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