Contaminant proteins showed higher than target protein as compared between CBB and immunoblotting (anti-B4GALNT1 mAb) in attached image, Please suggest to improve purity of target protein.
Mouse melanoma cell were transfected in 10 cm (2 dishes).
buffer were used:
Equilibration buffer (20 mM sodium phosphate and 0.5 M NaCl, pH 7.4)
Washing buffer (20 mM sodium phosphate, 0.5 M NaCl and 40 mM imidazole, pH 7.4): washed three times using 500 μl washing buffer in each
Elution buffer (20 mM sodium phosphate, 0.5 M NaCl and 500 mM imidazole, pH 7.4): twice elution- 1st eluted with 200 μl then again with 100 μl
Eluent was concentrated using Amicon ultra -4 centrifuge and final volume was 50 μl.
For western immunoblotting and CBB sample were applied 5 μl and 15 μl respectively.
I also tried using 20 mM imidazole in washing buffer but contamination was higher than used 40 mM imidazole.
Your Western blot shows that your protein is present in the Ni column sample, but it is not a major component. (It also shows 2 or 3 bands, which may mean you are getting some proteolyis during purification.) It is not surprising that a single Ni affinity chromatography step was insufficient to purify the protein if it is not very highly expressed. For one thing, it is well-known that mammalian cells normally contain some proteins that bind to Ni columns. You will need to use multiple purification steps. I suggest using ion exchange chromatography and/or gel filtration chromatography first, followed by Ni affinity chromatography.
Hi Robiul,
I would suggest trying to add some imidazole to your lysis and your equilibration buffers (maybe 20-30mM). Also, try to see if your protein does elute at a lower concentration than 500 mM imidazole. It is not necessary to go that high if your protein elutes very well for example at 350 mM.
As Adam suggested, you will perhaps want to run your samples through size exclusion column after all.
Good luck!
We purify our protein of interest on a column using a gradient. Try doing a stepwise elution from 20 mM imidazole and up. For instance, wash with 20mM, then elute and save fraction using 50, 100, 150, 200, 250, and 500 mM in a stepwise fashion. You should then have six fractions to examine by SDS-PAGE. See where maximum recovery of purified protein occurs.
I have never used the beads before. I agree, 500mM seems too high a concentration.
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