Recently I have been involved in this problem: After expressed by adding IPTG, lysed with B-PER, and resolved with Buffer B (8M urea), incubate with Ni-NTA beads, everything looks fine. When I elute with different pH values, I start with pH=6.3 (Buffer C) as a wash buffer (3 times), then elute with pH=5.9 (Buffer D) 3 times, then I elute with pH=4.5 (Buffer E) 5 times. All samples from 3 X C, 3 X D and 5 X E were performed with SDS-PAGE, and the results seem really good. The protein of my interest have a good expression quantity. During the SDS-PAGE, I will keep eluting several times (around 10, max to 20), then after verified with SDS-PAGE, I collect all solutions into dialysis bag and put into 1 X PBS. I do RT 1h, then replace with new PBS, 4℃ overnight, and replace with new PBS RT 1h+ on the other day. After this three-step dialysis, the bag was coated with PEG8000 powder to concentrate. I sucked all solution from the dialysis bag and test with Lowry BSA method and SDS-PAGE again to see the concentration. The result showed that the concentration is very low, consistent between Lowry and SDS-PAGE. I checked the dialysis bag, 6-8kD, the size is correct. Previously I tested the last tube I eluted with Buffer E (20th time) and the protein is still of good elution. I just have no idea where is the problem...
Dear Han
Why you had UREA8M (bufferB)?
Because if Urea is necessary for protein solubilization, it will induce protein unfolding and the dialysis in PBS will result on reduction or _UREA concentration will represent a sort of refolding step and is not strange that during the refolding you observe protein precipitation and reduction of the soluble protein.
Did you observe any precipitation during the dialysis?
best regards
Manuele
Manuele Martinelli Thanks for your comment! Yes, I saw pretty much precipitation after dialysis. I was thinking that there are precipitation at least means there are a lot protein inside, but come with like 0.2mg/mL from the Lowry assay. Buffer B was just follow the instruction of the protocol from Qiagen. Buffer B to resolve the insoluble protein and then use Buffer C, D, E to wash/elute.
Dear Han
if the B-per buffer is able to solubilitze our protein, (you can chjeck it by loadingthe b-per fraction on sds-page) then you can skip the use of buffer B and you can perform a Ni-nta purificationm with buffer whivhhich do not contain urea.
You can preapre the buffers by your self.
Generally for IMAC with ni-nta of soluble protiens > i'm using
binding buffer -- Tris 20mM, Nacl 300mM, imididazole 10mM pH=8
wash buffer --> Tris 20mM, Nacl300mM, imidazole 20mM pH=8
elute buffer --> Tris 20mM, NaCl300mM, imidalzole 300mM pH=8
in case that your protein is not soluble n B-per, than before use UREa, i suggest you to try different expressin strategy to improve its solubility if possible. However there is not a common solution valid for all proteins, each protein has is own properties and is difficult provide you reilable suggestions with out know more detail about your protein sequence.
ciao
Manuele
- Badges
- Science method