I am working with Recombinant His tag proteins with molecular size of 10 and 5 kDa, after the completion of gel run, i stained with coomasive blue dye .After few minutes of destaining i can see my expressed protein but after prolonged washing my expressed protein disappeared. i also tried to purify using Ni NTA beads but i did not get any thing,please suggest me the best way to purify recombinant his tag protein.Thanks
The prolonged washing may be vanishing because of two reasons: a) you are washing too much and b) you have not fixed your gel enough. Purification protocol are available from your gel manufacturer which work very well. For lower mol wt peptides/proteins you have to be extra careful during elution and washing.
You may need to fix these before you do the staining and washing. I have worked with small antimicrobial peptides about this size and had similar issues until I fixed them in aqueous glutaraldehyde (10%) for 1 hr
Hi there,
1. You have commaisse staining issues which can be fixed by silver staining which is irreversible staining .
2. Use High percentage gel with low pore size , do not let complete run out of the dye from SDS-PAGE since, low molecular weight will shoot out of the gel, Stop your run of the gel before the dye run out the sealing bottom of the gel.
2. His-tag protein purification trouble shoots:
1). Do a mini Induction at low volume with appropriate heterologous bacterial expression system with codon optimized one such as RIL or Codon Plus .cells.
2). Standardize the concentration of IPTG at which optimum protein get induced.
3) If there is a protein solubility problem , then induce at low temperature.
4) Regenerate Ni-NTA column and equilibriate and bind protein with 3mM imidalzole and wash with 25mM imidazole and elute with 300mM imidazole.
Use Silver staining to assess the protein profile in SDS-PAGE. SInce commassie would if you destaining adequately.
I hope this was useful.
Rajkumar
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