Hello, I try to purify my His-tagged protein with CM-Sepharose column, but my protein came out through the column when I washed it with 50 mM citric acid buffer without any salt. Does it mean that my protein not bind with the column? Should I try different columns or am I doing it wrong?
Here's the condition of my purification experiment:
- my protein pI is 5.11 and I use 50 mM citric acid buffer pH 6.5
- I use NaCl gradient of 0.2, 0.4, 0.6, 0.8, and 1 M
My protein size is around 38 kDa and I have lysed it with sonicator using PBS buffer followed by centrifugation at 6000 rpm for 15 minutes. My protein is in soluble form in the supernatant. I have sequenced my plasmid and the His-tag is there.
Dear Dininurilmi
If I remember well the CM is a cationic exchange resin that mean that is it able to bind protein dissolved in buffer with pH lower than their isoelettric point. In your case, If the protein pI is 5.11 and the pH of your buffer is 6.5, you need to use an anionic exchange resin as the DEAE or Q.
Just a question:
If your protein contains an his tag, why are you performing the anionic exchange instead the IMAC?
Is this the second step after IMAC?
Because in this case, if your protein sequence contain a protease digestion site (eg TEV or fatt.xa) suitable for His-tag removal, you can also try to perform subctractive IMAC.
If you are interested to know more information about it you can find a video tutorial on my blog ProteoCool
https://proteocool.blogspot.com/p/index.html
Ciao
Manuele
Dear Dininurilmi
If I remember well the CM is a cationic exchange resin that mean that is it able to bind protein dissolved in buffer with pH lower than their isoelettric point. In your case, If the protein pI is 5.11 and the pH of your buffer is 6.5, you need to use an anionic exchange resin as the DEAE or Q.
Just a question:
If your protein contains an his tag, why are you performing the anionic exchange instead the IMAC?
Is this the second step after IMAC?
Because in this case, if your protein sequence contain a protease digestion site (eg TEV or fatt.xa) suitable for His-tag removal, you can also try to perform subctractive IMAC.
If you are interested to know more information about it you can find a video tutorial on my blog ProteoCool
https://proteocool.blogspot.com/p/index.html
Ciao
Manuele
CM sepharose is a cation exchanger resin, your protein at pH = 6,5 is charged negatively, so you need to change your resin. Just the His tag is not enough to bind your protein to that resin.
Yes , you need to work at isoelectric point of your protein should be less than the pH used. Please check the link below will certainly help you to resolve the issue:
Article A unified method for purification of basic proteins
Just one further comment: Yes pI (theoretical or experimental) of a protein is an important tool in assessing a proteins charge under differing pH conditions. However, due to the very nature of proteins and distribution of both negative/positive charge across a proteins surface, it is entirely feasible that a protein with a pI of 5.11 could bind to a cation exchanger at pH 6.5. I would test the conductivity of your sample and equilibration buffer to ensure that the sample is lower. Although I agree that in this case the protein most likely does not bind to CM under conditions used. The best way is to test the binding of your protein of interest to both cation/anion exchangers over a broad pH range under small scale: 50 uL resin equilibrated with diff pH buffers then incubated with protein solution for 5 mins on orbital mixer. Spin and analyse supernatant for target (make sure that your protein is stable at pH tested and does not ppt)
I agree with Ian - sometimes you can get a protein to stick to the CM resin even if it doesn't obey the rule with the overall charge; if there is a significant patch of positively charged residues the protein could still bind to the resin. One thing that is very convenient with the cationic exchanger is that the majority of bacterial proteins won't bind to it so you get a really clean prep using only a short gradient. When switching to DEAE you will need to do a much shallower gradient to get rid of all the other stuff that will bind to the column. It is also likely that you would need a second run on a stronger anion exchanger such as MonoQ to get a clean protein.
In any case, it is reasonable run the IMAC first.
Good luck!
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