I produced some small secreted proteins (fungal effector proteins) with Pichia pastoris system and I have the supernatant samples (in BMMY media) to check for the expression. I am not sure if it is better to do acetone precipitation to concentrate the proteins and remove the media contents for better results of western blot? My proteins are his-tagged.
4/17/25
Dear Cindy,
You can concentrate your protein by precipitation as you mentioned. Acetone can be used for this purpose, although sometimes enzymes or other proteins are denatured by organic solvents. You can instead try using ammonium sulfate (AS). This has been used for decades as a (usually) mild/safe way to precipitate proteins w/o denaturation. You may have to try different concentrations of AS to determine what % AS saturation will precipitate your protein and leave unwanted media components in solution. The %AS saturation will vary from protein to protein.
What may work better for you is IMAC, or Immobilized Metal Affinity Chromatography (See attached.). This uses resins to which metal ions (e.g., Ni, Co, Fe, etc) are attached by chelation. This works very well w/ His-tagged proteins. The protein will bind to the metal (Ni works very w/ His-tagged proteins), while proteins w/o His are unbound and wash from the column. Your His-protein can then be eluted by using a gradient of imidazole which competes w/ His for the Ni ions on the resin. There is a lot of information in the literature on use of IMAC w/ Ni and His-tagged proteins.
I hope this information helps you.
Bill Colonna Dept. Food Science & Human Nutrition Iowa State University, Ames, Iowa, USA [email protected]
W.J. Colonna Hi thanks so much for your response. I saw in papers that they dialyze before purifying with a Ni-NTA column is it necessary to do that in advance?
5/4/25
Dear Cindy,
It's probably a good idea to dialyze the sample before the Ni-NTA step. This will give you a "cleaner" material to work with and will remove any low molecular weight substances in your preparation that might interfere w/ the chromatographic purification step.
If decide to do the dialysis, be sure to check the molecular weight cut off (MWCO) of the dialysis membrane. The MWCO tells you the size of the molecules that are retained. The membranes are available with different pore sizes. You mentioned that you are working with "small secreted proteins". You need to be sure that the pore size of the membrane you select is small (i.e., "tight") so that your proteins are retained while enabling the removal of low molec. wt (potentially interfering substances. If you use a membrane with a too large a pore size, your proteins might be lost during the dialysis.
I hope this information helps you. Good luck w/ your research!
Biill Colonna
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