5 replies to this topic

[PaddyS]

Hi all,

I am working on a GPCR which is expressed as an insoluble (not inclusion body) precipitate by cell-free protein synthesis.
My current work flow involves expression, resolubilisation, functional assays and/or purification. To date I have had no issues with resolubilisation (I get complete resolubilisation in <10m minutes at 30 degrees C with shaking).
I reolubilise into the detergent 14:0 lyso PG (LMPG) at 21mM (1%) link below. CMC is quite low, around 0.05mM
Unfortunately upon application to Ni-NTA I loose the majority (>90%) in the flow through. This is regardless of the pH (7.5 or 8) and so far regardless of the dilution of the detergent to 0.1mM by increasing the volume of the sample (eg: starting volume of 1mL made up to 200mL)

Ni-NTA binding buffer is 20mM NaPi pH7.5 or 8, 150mM NaCl, 5mM imidazole plus protein/detergent

What I do recover is a very small fraction and is unfortunately of no use to me at such small quantities.
Could someone please help me with any options that avoids complete denaturation.

http://avantilipids....atnumber=858120

Additional info: I wish to bind to Ni to perform detergent exchange with DDM

Cheers,

PaddyS

[mdfenko]

are you using enough ni-nta to bind the amount of protein you're loading?

is the ni-nta fresh or used and regenerated?

[klinmed]

I have not come across the detergent you are using before (but it is obviously fairly non-denaturating). In this regard, are you sure that the tag is exposed in the partially denatured protein? To explore this possibility, maybe you could look at the effect of adding a denaturant (urea/guanidine) and see if this improves binding.

[PaddyS]

Hi all,
Sorry for the delay.and thanks for the responses

@mdfenko The Ni-NTA, while not fresh has been only used once before and is regenerated.

@klinmed: I am worried also that the tag is not exposed enough to get good enough retention. But I wish to prevent and denaturation as I have shown previously that the receptors is somewhat folded and active and I may throw a spanner in the works by trying to do a refold.

Pat

[mdfenko]

you can try increasing the length of the his tag.

[Bluefunk]

Have you tried a different detergent? Triton?. Im assuming you havent tried removing the detergent, or ifyou do the protein precipitates? Youre a fair way above the CMC  soif you want to remove the detergent you'd have to do gel filtration as the micelles would be presumably larger than the dialysis MWCO