4 replies to this topic

[Megan Barker]

Hi there,

I have a secreted protein (95kDa) with a His tag, which I purify by batch-binding the (filter-cleared) culture supernatant to nickel beads. I let the batch-binding take place overnight, then leave the mixture still for a couple hours to allow the beads to settle. Pouring the media/beads tends to take quite awhile because the column clogs up to the extent of blocking all flow. These days, I usually 'resuspend' the clog, though this tends to bash up the beads a bit even if I do it gently. The beads seem to stick to centrifuge tubes so spinning down (even slowly) causes me to lose a lot of yield.

I was hoping that I could speed things up by putting my nickel beads into dialysis tubing (higher MWCO than my protein, say 300 MWCO) so that I don't have to pour and resuspend and wait (repeating ad nauseum for a 6L growth).

So my question: has anyone ever tried this? Will the Ni-NTA agarose beads stick to the dialysis tubing (which would be problematic for purification and re-use)? Does anyone foresee any other complications here?

Thanks in advance for the help,
megan
multanova@gmail.com

[T C]

Hey,

Its worth giving a try but the extent of binding may be lowered. How about diluting the supernatent and filtering it using a stericup and use it for binding?

Best,
TC

Megan Barker, on Oct 6 2009, 12:41 AM, said:

Hi there,

I have a secreted protein (95kDa) with a His tag, which I purify by batch-binding the (filter-cleared) culture supernatant to nickel beads. I let the batch-binding take place overnight, then leave the mixture still for a couple hours to allow the beads to settle. Pouring the media/beads tends to take quite awhile because the column clogs up to the extent of blocking all flow. These days, I usually 'resuspend' the clog, though this tends to bash up the beads a bit even if I do it gently. The beads seem to stick to centrifuge tubes so spinning down (even slowly) causes me to lose a lot of yield.

I was hoping that I could speed things up by putting my nickel beads into dialysis tubing (higher MWCO than my protein, say 300 MWCO) so that I don't have to pour and resuspend and wait (repeating ad nauseum for a 6L growth).

So my question: has anyone ever tried this? Will the Ni-NTA agarose beads stick to the dialysis tubing (which would be problematic for purification and re-use)? Does anyone foresee any other complications here?

Thanks in advance for the help,
megan
multanova@gmail.com

[swanny]

Megan Barker, on Oct 6 2009, 05:11 AM, said:

Hi there,

I have a secreted protein (95kDa) with a His tag, which I purify by batch-binding the (filter-cleared) culture supernatant to nickel beads. I let the batch-binding take place overnight, then leave the mixture still for a couple hours to allow the beads to settle. Pouring the media/beads tends to take quite awhile because the column clogs up to the extent of blocking all flow. These days, I usually 'resuspend' the clog, though this tends to bash up the beads a bit even if I do it gently. The beads seem to stick to centrifuge tubes so spinning down (even slowly) causes me to lose a lot of yield.

I was hoping that I could speed things up by putting my nickel beads into dialysis tubing (higher MWCO than my protein, say 300 MWCO) so that I don't have to pour and resuspend and wait (repeating ad nauseum for a 6L growth).

So my question: has anyone ever tried this? Will the Ni-NTA agarose beads stick to the dialysis tubing (which would be problematic for purification and re-use)? Does anyone foresee any other complications here?

Thanks in advance for the help,
megan
multanova@gmail.com

I've never thought of using dialysis tubing, but would not recommend it, because you will have to concentrate your protein from the large dialysate.

What volume of beads/ lysate are you working with? If it's not too large, there are a number of empty column bodies to use. You can place the slurry into spin-X tubes and gently centrifuge: one of those little bench-top nanofuges should do, max "g" <1000 x g.

[Megan Barker]

Hi guys,

Diluting will likely not work, because it's already at 6 L of culture. (this is supernatant, not lysate - because the protein is secreted.) I already do filter-clear the supernatant, but things seem to crash out afterwards when I run the column in the cold room.

This time around, I'm going to cool the supernatant to 4 degrees before filtering - in case it's just a temperature-saturation effect. Will let you know if it improves.

Also I talked to someone about the dialysis tubing idea - and they thought that the big problem would be the Kd - having things binding to the beads would create a upward concentration gradient which would reduce the amount that bound.

Anyhow, thanks!

megan

swanny, on Oct 7 2009, 01:13 AM, said:

Megan Barker, on Oct 6 2009, 05:11 AM, said:

Hi there,

I have a secreted protein (95kDa) with a His tag, which I purify by batch-binding the (filter-cleared) culture supernatant to nickel beads. I let the batch-binding take place overnight, then leave the mixture still for a couple hours to allow the beads to settle. Pouring the media/beads tends to take quite awhile because the column clogs up to the extent of blocking all flow. These days, I usually 'resuspend' the clog, though this tends to bash up the beads a bit even if I do it gently. The beads seem to stick to centrifuge tubes so spinning down (even slowly) causes me to lose a lot of yield.

I was hoping that I could speed things up by putting my nickel beads into dialysis tubing (higher MWCO than my protein, say 300 MWCO) so that I don't have to pour and resuspend and wait (repeating ad nauseum for a 6L growth).

So my question: has anyone ever tried this? Will the Ni-NTA agarose beads stick to the dialysis tubing (which would be problematic for purification and re-use)? Does anyone foresee any other complications here?

Thanks in advance for the help,
megan
multanova@gmail.com

I've never thought of using dialysis tubing, but would not recommend it, because you will have to concentrate your protein from the large dialysate.

What volume of beads/ lysate are you working with? If it's not too large, there are a number of empty column bodies to use. You can place the slurry into spin-X tubes and gently centrifuge: one of those little bench-top nanofuges should do, max "g" <1000 x g.

[swanny]

Hi Megan,
Can you do tranverse flow filtration/diafiltration? We used to take a couple of litres of medium down to ~30 ml of ion exchange binding buffer in half a day, so your 6 litres should be achievable in one day, even if you only get the volume down to a couple of hundred ml.