I am trying to purify a protein ( Kinase domain) , ~80 kD in size. I loaded the crude extract after expression on affinity column ( Ni-NTA) . On running SDS gel I got multiple contaminating bands in eluted fractions. Then I tried loading the eluted fractions on FPLC size exclusion column ( Superdex 200), but inspite of the contaminating bands being of low molecular weight, I am still getting them with my protein band on SDS gel.
* Protein has 13 cysteine residues.
Can anyone help me out with this problem or is it due to hydrophobic linkage of my protein with other contaminating proteins that remains bound and comes out at the same time after size exclusion chromatography?
It's possible that the protein has become crosslinked to other proteins by disufide bonding if the extract was not prepared and maintained in a reduced state. But it is also possible that you just need to add another purification step. I suggest adding an ion exchange chromatography or hydrophobic interaction chromatography step to the purification.
Hi there,
It might be that your purified protein is partially cleaved and still remaining in native conformation explaining the observation of several lighter bands under denaturation condition only.
If the band intensity of the contamination bands, is very high, excise them and do trypsin in-gel digestion and confirm their identity. If they are bacterial His rich proteins, u can give more washes with imidazole from 40 to 70 mM.
If they are chaperones, then your wash buffer should contain 10 mM Imidazole,1 M NaCl, 20 % glycerol, 5 mM MgCl2 and 5 mM ATP. Sometimes 40-50 column washes is required.
All the best.
Hello,
Before you put your protein in column, make a bond with the afinity resin in a plastic tub in cold camera 4C. Later wash at least three times in a very slow rotatory stiring, with forty times more buffer than resin and protein, for 30 minutes at time. Finely load your protein and resin in the column. Wash more one time in the FPLC or HPLC and latter perform the eluition step with your eluition buffer. Your protein will became prety clean. If you want improve, because some heat shock proteins can bind to your protein , put a little bit of triton or tween-20 only 10 microliter in the first washing. Then load in the colun. If you do that, your protein will became very clean, most than 98% in purity.
Tell-me later if your ptotein became enough clean.
Good Luck.
Hi,
Before you perform Ni-affinity chromatography, try performing ion exchange chromatography on your crude extract. This would eliminate some contaminants and will also leave you with much cleaner sample for Ni-NTA chromatography. When you perform Ni-NTA, try increasing the imidazole concentration gradually to elute off contaminants in lower imidazole concentration. Hope this works for you! Good luck!
Other than all the optimisation of purification such as increasing the level of reducing agents, changing the elution conditions, add more steps, etc etc. Do you think you have any antibodies (other than anti-His) to detect your protein by western? This you can be sure that the band(s) are indeed your protein, not some remaining junks from E.coli?
Hi, Exactly, your protein is forming a disulphide bond/cross link with other E. coli proteins or your protein itself is oligomerising. I would do a western blotting to check the oligomeric state. You need to add 1 or 2mM DTT in your binding buffer as well as in the gel filtration buffer. But be care full some of the NiNTA beads are not compatible with DTT, some are OK up to 2mM DTT. Check that!!
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