Hi,
We have encountered something strange. Our group is purifying an N-tagged 6xhis protein with Talon 5ml prepackaged column. When we first tried with 2ml sample volume, everything worked great. Then we tried 8ml sample volume (4x), and we expected a 4 fold increase in protein yield. Indeed the result was as what we thought.
However, when we apply 15ml sample volume, the protein yield is about the same as what we get with 8ml sample volume... It was about 1.5mg of protein, Theoretically, Talon column is able to bind 20mg protein per ml resin, so there is no way we were reaching that limit.
Our binding buffer: 50mM PBS, 300mM NaCl, which was used as cell lysis buffer with additional PMSF.
Wash buffer: 50mM PBS, 300mM NaCl, 5mM Imidazole
Elution buffer: 50mM PBS, 300mM NaCl, 150mM Imidazole
No EDTA was involved at all. And we don't think it's the buffer issue otherwise it would not work with 2ml and 8ml sample volume, right?
Please help! Thank you so much!
PS we really don't want to purify under denatured condition, so let's just skip the detergent part! :)
Protein yield vary from prep to prep. Your buffer is fine. For elution better use 250 mM Imadizole. If your protein is not binding to column, it must be in the flow through, isn't it. You can reload that after stripping the talon beads with 500 mM Imidazole and requilibrating with your binding buffer. Suggestion.......Do affinity manually with loose talon beads (i always do that). Run all the fraction supernatant, pellet flow through, wash and elution on sds-page and see where ur protein is going.
If you didn't produce all 3 samples (2 ml, 8 ml and 15 ml) at the same time and from the same culture, then there is always going to be same difference between them.
Did you check the flow-through of the 15 ml sample to see if it contained un-bound His-tagged protein? If it didn't, then the sample had a lower protein concentration than the other samples and you'll need to play with your culture conditions to improve the yield of larger samples. If it did contain un-bound His-tagged protein then your binding protocols needs to be optimised. I haven't used TALON columns so far, but I think you probably need to improve your buffers a bit.
At 50 mM, your PBS will have very little buffering capacity and the cell lysate can easily alter the overall pH of the sample after lysis (cell lysate can often be more acidic than you think), that's why I always recommend using buffers at 100 mM to make sure the solution stays at a pH > 7.6. The binding affinity of His-tags to the columns matrix is reduced at pH < 7.6 in the case of Ni-NTA ... I think TALON (Co-NTA) is more forgiving in this regard, but you still should try to have the pH > 7.
Also, if the eluate from the 15 ml sample was quite dirty, I would recommend adding some imidazole to the lysis buffer to reduce non-specific binding of other proteins to the column, which reduces the amount of your target protein bound to the column (this might also explain why you had less yield from the larger sample). For Ni-NTA this varies from 10 to 30 mM imidazole and depends on the particular affinity of different His-tagged protein constructs. From the GE TALON manual it seems that 2-5 mM imidazole could be included in the lysis buffer.
If these recommendations don't improve your yield, you can always grow larger cultures and then use two TALON columns in tandem (you simply screw one 5 ml column into a second one and then your cell lysate runs over 10 ml of TALON matrix instead of 5 ml, which should be enough to bind all your His-tagged protein from a 150-200 ml sample ... if it has the same concentration of your protein as your current 15 ml sample). Remember that you'll need to double the volumes of the wash and elution steps in this case.
Final thought, maybe the column you used had been run quite a few times before and simply needs recharging (I routinely strip and recharge my Ni-NTA columns after every third run ... though TALON columns might be different in this respect).
I hope this helped.
Hi Peter,
Like what the others said, protein yield varies from prep to prep. For one of my proteins, I went from 200 ml to 2 l, and still only got very little increase in the yield. What are the volumes of your culture? As you go up in volume in a fixed container, the surface area to volume ratio decreases, and it affects the aeration. Grow your cultures in a number of small flasks instead of one big one would help.
Best regards,
Arthur
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