In most protocols, 50% methanol is used for protein A regeneration, followed by storage, usually in water/buffer/20% ethanol.
In my experience, it is critical to apply a 50% methanol wash so the protein A resin can be reused many times. I assumed it's because 50% methanol clears out most salts and organics. But is there a specific function of the 50% methanol or reason that it is used other than cleaning / antibacterial?
Thanks.
Dear Leran Mao
to my knowledge the modern protocol used for proteinA regeneration are based on NAOH washing. It is true that native protA is not so stable and binding efficience is reduced after several time using this regeneration approach and for this companies (as Cityva) developed some new protA stabilized able to resist to alkaline pH
as the MabSelect@
https://www.cytivalifesciences.com/en/us/about-us/our-brands/mabselect
that can be regenerate several times with NaOH.
https://proteocool.blogspot.com/2022/07/tips-of-week18-mab-select-cheaper.html
best regards
Manuele
Hi,
Thanks for both answers - I assume methanol is not used at 100% because it would cause salt to precipitate and clog the column.
Manuele Martinelli
Interesting - I used to use 0.1M NaOH for cleaning but only once in a dozen runs or so. Most of what I read suggested using 50% methanol for more routine cleaning because it's less disruptive. And so for some reason I thought there's another function of 50% methanol for 'regeneration' - like it could rehydrate the resin or increase protein stability.
Your blog was very cool, keep up the great work!
DEar Leran Mao
with MAbselect was are performing NaOH washing at the end of each purification.
I dont know shy the methanol was used, it just because do not affect protA stability or because it is really better. However is it possible that this coming just from the fact that methanol is largelly expoited as washing agent in
Standard HPLC coloumns (were is used to can wash away residual strong polar impurities, and hexanes reactivate the surface of the silica gel) and this protocol extensively used in organic chemistry was automatically tested on coloumns for protein purification and therefore also in protA.
However protein are little different and more complex that organic molecules. For example, in my blog you can find also a post (Pills#22) showing as, differently to what suggested from Malvern, that suggest to use methanol and HNO3 for cleaning of LALS/RALS OMNISEC detector, in case of proteins only the NaOH (and little the SDS) are really able to clean well the cells and restore good bakground signals.
thanks for the comment about the blog.
It is something that i'm doing in my free time, i know that the graphic lyout is quite old style and not appealing but i'm happy if it could be useful to someone working on protein chemistry.
best
Manuele
- Badges
- Science topic
- Similar topics
- Chemistry
- Organic Chemistry