Hello everyone,
I am trying to purify my protein (enzyme) expressed in Pichia pastoris through ion exchange chromatography. The protein is extracellular, so I extract the supernatant after centrifuging and discarding the pellet, then I filter the sample and dilute with 40mM sodium acetate Buffer. The elution makes with 40mM Na acetate and 1M NaCl.
When I make the SDS-PAGE I have the following samples: Crude stratum (supernatant), Not retained and elution peaks. In Western Blot appears only the fraction of the crude and a very weak band of the unretained, but the peaks of the eluates nothing. Before applying to the SDS Gel I precipitate all samples with 10% TCA in acetone. After washing the proteins with acetone I dry it and get a white pellet that dilutes directly with the sample buffer to apply on the gel.
It is important to mention that after diluting the pellets in the sample buffer some samples turn yellow (especially the fractions eluted) but I stabilize the pH with Tris 1M and they get blue again. Even though the results are negative.
If someone has experience with it or went through a similar difficulty, please write to me. Thank you!
Is the amount of the protein in the unretained fraction sufficient to account for all the protein that was applied to the column? Check this by comparing the band on the Western of the unretained fraction to the band in various amounts of the applied sample.
If not, then the protein either stuck to the column so tightly that it was not eluted by 1 M NaCl, or it became degraded, or it precipitated and is stuck in the column. For the first possibility, a change in pH could help reduce the affinity of the protein for the column. Include protease inhibitors in the sample to reduce proteolytic degradation.
Do you elute with a salt gradient or just a 1M NaCl step. If it is a step elution, the exposure to 1M NaCl may cause the protein to aggregate/precipitate and get stuck in the column. Use a gradient elution instead. Or switch to a less chaotropic salt, such as ammonium sulfate.
Consider other methods of purification. Ammonium sulfate fractionation is a good starting point for a crude culture supernatant. Gel filtration chromatography is a good follow-on to ammonium sulfate fractionation, followed by ion exchange chromatography.
Thank you for your recommendations. I think the unretained fraction is sufficient to account, yes, because it is weak but only because it is diluted in the buffer. At the time of the elution in the graphic appears a very high peak but without my protein.
Thank you very much, mister Adam.
So the protein never bound to the column. This could be because the solution has too high an ionic strength (salt), or because of a poor choice of pH, or because the protein simply lacks the necessary charged residues on its surface.
One other hint: When preparing the samples for SDS-PAGE, use volumes equivalent to the original lysate. For example, if you had 10 ml of lysate and use 1 µl for electrophoresis, and if you had 1 ml of eluate from the column, then use 0.1 µl of that for electrophoresis. That way you can directly compare band intensities to see where your protein of interest happens to be.
The other thing you should do is to also take samples from everything, so for example also from the flow-through of columns. in each case, determine total protein, band intensity in PAGE, and enzymatic activity, both concentration (per ml) and total amount (concentration times total volume). This allows you to see where losses occur.
It appears that your protein of interest is in the unretained/non-binding fraction. Therefore it is necessary to adjust the pH of your supernatant sample and buffer solutions (both of the equilibration (buffer A) and elution (buffer B) buffers). Increase the pH to above the pI of the enzyme of interest for anion exchange chromatography or decrease to below the pI for cation exchange chromatography. Ideally, the sample should be dialysed in the low salt buffer (buffer A) but as you are working with the supernatant this may be difficult, try and concentrate first (e.g. ammonium sulphate precipitation or ultrafiltration)
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