I have been trying to purify a protein through cation exchange chromatography on a Nuvia Q column (BioRad). For some reason when I measure the protein content (through ELISA) in the flow throgh, washing, elution and stripping of the column and I add the together, I only recover no more than 60% of what I loaded. I wonder where is the 40% left over.
I also tried to add some arginine, as a stabilizer, but it didn't work as arginine increased the conductivity and I collected almost everything in the flow through.
Does anyone have an idea on how I can solve this problem?
Thank you.
Andrea
What is the pI of your protein? What buffer are you using for adsorption?
I am using 20mM phosphate buffer pH 7.0 and the pI is around 5.2. The protein is eluted with 0.15M of NaCl.
First, I suggest you measure your protein concentration with the BCA protein assay kit or other protein assays instead of ELISA. The problem by measuring with ELISA are many but it may be providing some information. You may have a higher amount of protein recovered than what the ELISA says, however the protein may have changed conformation in the column and the antibodies used do not recognize the non-native form.
Another thing to keep in mind is the concentration of protein. The less you load the more you lose in ion exchange columns. And, since you are measuring the amount of protein flowthrough etc- it should add up to what you have loaded- although some is always lost in the walls of vessels (glass, plastic tubes etc). If you are dealing with a hydrophobic or membrane protein, make sure to 'strip' the column with a buffer containing urea or other reagent that will solubilize these proteins and thus account for the missing protein.
If you are dealing with a membrane protein you must consider having a nonionic surfactant
Thank you very much for your answers.
@ Gustavo: unfortunately I am dealing with the first column of four chromatographic steps, so still a lot of impurities and that's why I can't use BCA protein assay. The protein I am purifying is hydrophobic but not a membrane protein and the ELISA I use recognize only the portion of protein that is still active (it's actually an enzyme).
At the moment my feedstock has a concentration ranging from 0.5 to 1mg/mL of protein, and after elution with 0.15M NaCl the column is washed first with 0.5M NaCl and then stripped with 0.5M NaOH.
@ Omar: besides phosphate buffer I have also tried Tris and histidine with equal results.
Thanks again.
If your protein is hydrophobic, as you state, it is very possible that neither NaCl or NaOH are going to dislodge bound protein. The hydrophobic protein may have come out of solution during the run and is still insoluble in the resin. To avoid losing some of the protein during the run, add a small concentration of detergent (e.g. SDS or an anionic one) a small amount of urea (2 M) may also help. similarly to clean out the column, you can run 6 M urea or a detergent to remove precipitate protein that will not be removed by salt or base alone.
- Badges
- Science topic
- Similar topics
- Engineering
- Petroleum Engineering
- Gas