Hi all,
I am working with a protein that I have characterized quite well by SEC using superose6 resin. In the last 2 purifications, I have been unable to recover the protein in any fractions collected, from the large (1.2 mDa complex) size, to the monomeric size 50 kDa, or anywhere in between or the void volume. Last time I ran this protein without successful purification, upon finishing my work, I equilibrated the column out of my buffer into H2O. At this point, I noticed a large peak on the trace right in the point that the conductivity was changing from my buffered condition, into H2O. I have not confirmed that this peak was my protein, however, it seems odd that my input is full of my protein, and the output of the run contains no trace at all. Is it possible that my superose6 is acting like a IEX column and that my protein is now binding in the presence of high salt and eluting in low salt? Has anyone ever experienced sticky proteins in their SEC columns?
Any advice is greatly appreciated!
Kyle
FYI:
- Buffer conditions - 25 mM Tris HCl pH 8, 300 mM NaCl, 2% glycerol
- The complex after purification with Ni-NTA looks typical under TEM with the characteristic 1.2 mDa complex
- Our column was subjected to a lengthy storage session in between the past successful runs and my most recent attempts due to our fridge breaking and the FPLC being out of commission
- The column has been washed with 6M Guanidine HCl, 1 M NaCl, 0.5 mM NaOH, and 15% EtOH in between the first failed attempt and the second failed attempt.
- Buffer stocks were not the same from previous runs and current runs. I was not the manufacturer of all of the buffer stocks either so there is a chance they could be different.. However, other lab members buffers (with fairly high NaCl concentrations 150 mM) do not elute the protein either
Hi,
What about the same fractions depending on the successful SEC experiments? Do they have no proteins on SDS-PAGE as well ?
Best
Hi there,
According to what you said (adsorption at high ionic strength, elution with water), it would be hydrophobic interaction chromatography rather than IEC. If the protein is not perfectly folded it could expose some hydrophobic patches and therefore stick to the column matrix...
Did you perhaps put the column under too much pressure (e.g., used a too high flow rat, esp. with viscous solution) and the resin beads collapsed If yes, you may have to purchase new resin or a new column?
You can test this with other proteins. For instance, when you run a protein size calibration mixture, do you see peaks and does the chromatogram make sense?
It is better, before doing FPLC, you should measure the protein concentration and do SDS PAGE to see if your target protein in the sample or not.
Also, you should do dialysis for it to remove imidazole after Ni-NTA Column, and do filtration (0.45 um). To make sure it is not aggregate inside the SEC column.
Normally, the large proteins precipitate when using centrifuge after the process of the sonicator, it is better to use "B-PER Complete Bacterial Protein Extraction Reagent" instead of the sonicator process. Catalog number: 89821.
https://www.thermofisher.com/order/catalog/product/89821.
According to your description I suspect that your target protein non-specifically binds to the resin because it is not completely folded, so that it cannot be eluted. In addition, I noticed that your buffer contains glycerol, which will increase the column pressure, so please check whether the column pressure is within the normal range firstly. If you have used Guanidine HCl/NaOH/Trypsin to wash the column, but the column still does not work properly, it is recommended that you replace it with a new one.
Hope it helps!
We have several proteins that behave like that - binding to resin and being released upon water injection. I agree, the problem can be partial unfolding of your protein. However, our proteins stay completely folded and functional afterwards, so you may as well just test the eluted fraction (activity, folding, temperature stability?) and maybe use that as a purification step, if you are lucky enough to have functional complex afterwards.
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