I am currently expressing a recombinant protein through the transduction of my plasmid into HEK 293T cells. Historically, I've encountered minimal issues and achieved high yields of protein at a satisfactory concentration. However, I've recently faced challenges as the protein expression has significantly decreased. Alongside investigating the underlying cause, I am interested in concentrating the protein obtained from my recent experiments. I am considering utilizing the Amicon Ultra for this purpose. However, I require clarification on the protocol specifics. Which buffers are recommended for this procedure? Additionally, I seek guidance on the recovery process for the concentrated protein. Your insights would be greatly appreciated. Thank you!
Dear Sergi Betriu Méndez
the use of amicon ultra is quite simple.
You can equilibrate the amicon ultra with the buffer in which your protein is stored and stable, add the protein, centrifuged and recover the concentrated protein from inside. For small volume amicon, eg 0.5ml, you can recover the protein by inverting upside down the membrane in an empty tube and centrifuging at low speed (e.g 1000g) while for higher volumes, eg amicon ultra-4 or amicon-ultra 15 you can directly recover the protein from inside using a gilson pipette or similar (200ul tips is suggested, since 1000ul tips is to big)
In the following link of my blog, you can find some tips about a possible mistake that you can do in ultrafiltration
https://proteocool.blogspot.com/2021/04/tips-of-week-1-11042021-overload-of.html
best
Manuele
Dear Manuele Martinelli
Thanks a lot for your answer and for your tips.
Best,
Sergi Betriu
Manuele Martinelli does your protocol work with pembrolizumab ? I want to remove excipients for post radiolabeling reactions.
Thanks for your blog.
Dear Laetitia Mistico
which is the MW of the exciptients used for radiolabelling reaction?
if are only small MW molecules with MW much lower than membrane cutt-off you can certanilly do it by performing multiple concentration-dilution cicles. A possible faster and less stressfull (for the mab) alternative is also the desalting which is normally used as last step of thermo commercial antibody labelling (with alexafluor or biotin) kits.
you can find more information about desalting on the following link on my blog, proteoCool
https://www.blogger.com/video.g?token=AD6v5dzeX21U_Er6jBChQIhE5zWtQhNjRjNXRPR7qk7KfWWSQxhf8PbUn4R6KLL0pn_7tU92M0-B3kEUHO9IkTpelmdO79QR53uSg29ZFV3j5D-siHAamTu1y2wOEQ8S4As_wpxzLvA
https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
good luck
Manuele
Dear Manuele Martinelli , the excipient are L-histidine Chlorhydrate de L-histidine monohydraté, Saccharose and Polysorbate-80 (E433). I think multiple concentration-dilution cycles are ok.
Thank you for your help
Laetitia
Dear Laetitia Mistico
i'm a fun of desalting, therefore i suggest you to try it, but i'm sure that also several dilution concentration steps will be ok. Just pay attention to do not concentrate to much your protein, since to high concentration may induce unwanted aggregation.
best
Manu
Manuele Martinelli Thank you.
I appreciate your help so much.
Laetitia
Manuele Martinelli your blog content is excellent. Thanks for sharing!
best,
Mariana
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