Hi Researchers,
Following to my previous questions, few of my chimeras are unable to secrete into media as expected but present in cells. Current plan is to purify from cells and I tried once with RIPA buffer it was good but not much protein I can see on elation fraction (Talon purification). I also seen some protein in flowthrough alternatively looking for better lysis buffer. It would be great if some one share your expert ideas to recover the pure product
Thank you in advance
Cells : Sf9 insect
Raju
This should help you retrieve the protein in elutions.
You can use 10mM Imidazole (pH 7.0) as wash buffer and elute with (NaCl 100mM, Tris 25mM (pH 7.5), 2-mercaptoethanol 5mM, imidazole 400mM (pH 7.0)).
Amit's suggestion would work really well for a standard nickel column (especially a good quality one like HisTrap), but...
If you plan to use TALON for purifying a 6*His protein, then it is essential that your binding/wash buffers DON'T contain any imidazole. TALON is great, and one of the reasons is that the concentration of imidazole required to elute the protein is much lower (normally the sort of concentrations used in nickel wash buffers), usually not enough to cause sensitive proteins to precipitate. I learnt this the hard way! Check your column to see the right concentration, and whether the column is sensitive to reducing agents like mercaptoethanol.
Some proteins are also quite sensitive to the pH of the elution buffer. In general, proteins appear to better tolerate slightly alkaline pH (8-9, sometimes even a touch higher). Remember that most buffers shift upwards in pH as the temperature reduces, so if you are working on ice, then a buffer that is set at room temperature will be more alkaline. With Tris, the change is 0.04 pH units per degree C, so this can be almost a pH unit of difference.
Good luck!
- Badges
- Science method