Dear Eniola Soetan

just some questions:

1) can please share with us some more information about the composition of your binding, washing and elution buffer?

2) Do you have a 6X his or 10x his tag?

3) What do you meand for 30 washes? 30 coloumn volumes?

4) Can please share the image of SDS-page to undestand the level of impurity that you obtain? Take in consideration that 1 imac step never provide high purity level, generally a second step (e.g SEC) is required to obtain high purities!

best regards

Manuele

Dear Eniola Soetan,

I used the strep tag which I find to be very reliable. It is a bit more expensive, but shows nice specificity and yields high amount of protein in the elution. However, it requires cloning of a new construct and the tag is a bit larger (but not by a lot).

This said, I would also check other potential problems.

1. Is the protein expressed properly (Do you see the correct band in SDS gel after expression)?

2. Is the His tag available on the surface (you can get an Idea by folding the protein in AlphaFold)?

3. How much protein is lost during the washing steps? An example of your SDS page would be helpful.

Best,

Tim

No matter what the manufacturers say, you will always have some impurities after elution. You need to share the protocol you are using for people to help you. 30+ wash range? Nobody will understand that.

Thank you for your answers Manuele Martinelli, Tim Habenicht and apologies for taking a while to get back to you! The his tag we are using is a 6x his tag. Our wash buffer is 10 mM imidazole in binding buffer and our elution buffer is 400 mM imidazole in binding buffer. We were doing washes of about 5-10 mL intervals and washing 30+ times doing that. Our binding buffer is composed of tris-HCL, NaCl, EDTA, and leupeptanin, aproptonin, pepstatin, and PMSF for protease inhibitors. It might be worth adding that for the last protein we purified, we switched to 25 mL washes since we had an idea of the expected wash range, and only ended up needing 50 mL worth of wash buffer before the Bradford's reagent assay actually turned brown. For all of the proteins, we have seen the brand expressed in the elution lanes to varying degrees of expression (we have been running gels with wells dedicated to each elution that was done and our elution washes have also been in the 5-10 mL range), but have also seen some expression in the wash lanes (although less than the elution lanes). After thinking about it some more, I realized that it's highly likely that the nickel beads were disturbed at certain points during the washes which may have caused some protein to flow through without making contact with the nickel beads and would also explai why some expression is being seen in the wash lanes of our gels. We are doing further purification of our proteins with other methods in any case. Thank you for your thoughtful responses, I appreciate it!