I am performing protein purification on an ÄKTA Prime Plus system. After His-tag affinity purification, I cleave the His-tag from my protein with thrombin. In the post-His-tag removal step, I reload the cleavage mixture onto a Ni²⁺ column to separate the cleaved (tag-less) target protein from the His-tagged contaminants (His-tag peptide, uncleaved protein, and His-tagged protease).
Since the cleaved target protein no longer binds to the Ni column, I understand that it should appear in the flow-through. My confusion is about which flow-through to collect:
- Should I collect only the sample-loading flow-through (while the cleavage mixture is being applied)?
- Or should I also collect the early wash fractions (immediately after the load, during Buffer A wash)?
- In practice, do researchers usually combine the load flow-through and the first few wash fractions as the product pool?
I would greatly appreciate guidance on best practices for fraction collection in this step.
In the first try, You need to collect all they fractions and evaluate them by WB or ELISA.
Take into acount that if You are injecting a lower volume of protein than your column volume, the protein gets traped in column until first wash, and the sample loanding elution is the equilibration buffer. Also some proteins sticks to the resin, so is important to check in wich time you get they protein.
Dear Imtiaj Uddin
you can collect both sample-loading flow-through and early wash fractions if you are performing a first was with a LOADING buffer to be sure that you recover all the protein loaded into the column.
As Jahaziel Gasperin Bulbarela suggest to you, the best is collect the fractions and evalutate it. SDS-page could be enough instead WB or ELISA if you are purifing mg scale of protein;
In the following link a video on my blog, about subctractive IMAC
https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz
best
Manuele
I am now clear. Thank you very much. Manuele Martinelli Jahaziel Gasperin Bulbarela
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining