Hello everyone, I am working on a 73 kDa protein with a theoretical pI of 5.28 and encountering an issue after dialysis. I performed purification using 20 mM Tris buffer (pH 7.8) and eluted the protein with 150 mM imidazole.
Immediately after purification, I set up the sample for dialysis without adding any glycerol. Post-dialysis, I observed a 3–4 fold increase in protein concentration and significantly thicker bands on SDS-PAGE compared to the pre-dialysis sample.
Although the solution appeared clear visually, centrifugation at 13,000 rpm for 10 minutes revealed a tiny pellet at the bottom of the tube. Additionally, I noticed an approximate volume loss of 150 µL after dialysis.
I would highly appreciate any insights or suggestions for troubleshooting this issue.
Thank you very much for your support!
There isn't really an issue. The concentration of water will establish if it goes out or in of the protein solution. If it goes out, protein concentration will increase, if the protein does not "like" this, it could precipitate (at least some of it).
Dear Mohd Javed
It is strange that you observe increase in protein concentration and at the same time the formation of a tiny pellet at the bottom of the tube, since generally at sample precipiration during dialisys may correspond a reduction in the concentration of the soluble protein and not the opposite.
Just to undertand better:
You notivces a volume reduction of 150ul after dialisys. Which was the sample starting volume?
Did you observed any opalescence of the sample after dialisys that may affect your protein quantification (expecially if you are perfroming it at 280nm)?
best regards.
Manuele
Manuele Martinelli Thank you, Sir, for your insightful comments. I also found it unusual that the protein concentration increased unexpectedly, which prompted me to centrifuge the sample at 13,000 rpm for 10 minutes to check for any kind of precipitation or aggregation. After centrifugation, I observed a tiny pellet at the bottom of the tube. I did not notice any opalescence in the sample. I am using the BCA assay for protein quantification at 562 nm, and the supernatant protein concentration remains consistent. Would it be acceptable to proceed with using this supernatant for my further experiments, such as Circular Dichroism (CD) or fluorescence spectroscopy?"
You can calculate the total amount of protein by measuring the initial and final protein concentration and volume, and then calculate the difference from these measurements. The amount should be the same, considering the experimental error, or less if the pellet is protein; otherwise, your pellet may not be protein, but rather some of the components of the buffers you use in dialysis that change their solubility. You can also dissolve the pellet and identify whether it is protein or not.
Francisco Pérez-pomares thank you sir for your reply, "After centrifugation of the sample, SDS-PAGE was performed on both the supernatant and pellet fractions. Protein bands were observed in both however, the band intensity in the supernatant was approximately three times higher than that in the pellet. I am considering using the supernatant protein for further experiment such as Circular Dichroism (CD) and fluorescence spectroscopy. Can I use it?
Long time ago I had similar problems, I do not exactly remember what I did. I remember that in my case, he pellet did not solve quite good and have distortion in the lane of the electrophoresis (I had the added difficut of working with halophilic proteins, and salts made it a little more difficult) Also I could not calculate the protein amount in the pellet since direct calculation was not enough accurate....
I thought then, that a liitle of protein precipitated at the same time than other components of the buffers (the answer I gave you) but still do not know exactly what happened....
Of course, I think that it does not affect at all to the protein in the supernatant... in fact you have probed it electrophoretically (even the nature of the protein in the pellet, so you may use both for futher studies... I suppose...) Regards
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