I am trying to purify the galactosaminyl transferrase, LgtD. I used BL21 E coli strain, lysis buffer (EDTA+NaCl+Tris Hcl) and washing buffer (imidazole conc 5 mM) and Elution buffer (20mM imidazole) all the buffer at pH 7, I am getting many other bands along with my POI bands with almost same intensity in the SDS Page. In western blot I have got the band of the protein, but it shows very light band, which shows very low yield. Please give me suggestion.
This depends on quite a few factors. Perhaps your low yield is due to poor expression. I also note that 20mM imidazole is not particularly high, I would probably try elution at a higher imidazole concentration.
As for the other bands, his purification can sometimes be quite dirty. I usually opt for alternative affinity tags. I am not sure what salt concentration you are using, but it's also usually recommended to use high salt concentrations during purification (between 300-500mM NaCl) to try and reduce ionic interactions with the resin, which might be a source of contamination. You can of course try further purification steps to improve the purity. Ion exchange, or size exclusion.
Hi Sayan,
I agree with James reply, 20mM imidazole is quite low for what I would typically use for an elution buffer - I am not surprised you are seeing other proteins in your gel. Are you sure you are fully eluting all bound protein, bear in mind a proportion of your tagged protein may not bind to the resin and will be washed away with the the other proteins - perhaps this is what you are seeing ? As James suggested perhaps try a higher concentration of Imidazole, after your 20 mM elution (250/ 500mM perhaps) to see if any POI remains bound. If your His-tagged protein does elute at 20 mM then I would suggest changing the position of the tag or use an alternative - I have had good success with strep tags for example.
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