I am trying to extract N-glycosylated proteins starting from 10 g of frozen plant lmaterial but I am facing a real problem with phenolics and oxidations. The protein solution is very dark brown, in color after centrifugation . In fact, I am using the following buffer in my extraction procedure: 25 mM potassium phosphate buffer + 0.5 M NaCl, 20 mM DTT , 10 mM TCEP, 0.1/1g PVPP and 1/200 protease inhibitor cocktail. so any help? in order to proceed for ConA column chromatography.
Thanks a lot.
Hello, Hasan Kolkas You can choose phase difference method like use DMSO or any organic solvent where your phenolics may dissolve and protein will be separated in water soluble phase. Even an prepartive HPLC also can be good option to separate them more accurately. Also KDS precipitation could be useful see link for more info.
Good luck! Article Red wine proteins: Two dimensional (2-D) electrophoresis and...
You can try using Ammonium sulfate fractionation. AS may require addition of PEG (suggested in some literature). You will need to have a desalting step afterwards though.
Dominique Anderson Thanks a lot dominique. i tried this option before where after AS precipitation the pellet was dark brown in color and again after desalting step i get a green protein elution extract in color. where i think the phenolics are still sticked to my proteins.
Karen A. Darbinyan okay good idea. i will try to mange this soon. thanks.
Hasan Kolkas to test if the phenolics are indeed adhering to the protein, you can try denaturing the proteins. You would need to refold and that has it's own issues in terms of protein function. You could look at an ethanol precipitation as well.
I also found this thread which may help you.
https://www.researchgate.net/post/Does_anyone_know_the_cause_of_plant_protein_precipitates_coloration_and_how_to_get_rid_of_those_colors
You can add ascorbic acid and free cysteine at about 1 g/liter. Also I would suggest to increase the amount of PVPP to 0,5 - 1 g/g plant material.
What is the material? and why include both DTT and TCEP?
Also I would suggest using another capture step than ConA. ConA is difficult to clean and it may be better to sacrifice an ion exchange column to get rid of the phenolics there. I have had good results with Capto Adhere (mixed mode anion exchanger) as a capture step which also binds the phenolics really well.
And remember that ConA really needs Ca2+ and Mn2+ ions at all times. Be sure to include 1 mM of each in all buffers and even in the sample. Otherwise you will get an extra 26 kDa band on you gels......
Be careful when adding free cysteine. You could get cysteinylation of free thiols in proteins.
Bo Pontoppidan thanks a lot. it was a nice idea i will try to read more about this capto Adhere. it is marchantia and iam adding both TCEP and DTT as a recommendation from a biochemist to avoid oxidation as much as we can. but i don't know if we switch to Capto Adhere we would be loosing material (proteins)?. and the addition of ascorbic acid may affect the pH of buffer? and what about free cysteine role?. regards
Capto Adhere is a multimodal resin, i.e., it has hydrophobic and anion exchange characteristics. To properly develop a step with this resin, you will need DOE software, as a OFAT (one factor at a time) will not get you too far.
Cysteinylation may be preferable to inter-protein S-S bridges.
I agree that Capto Adhere may require some work to optimize, but I have used it in several cases without DOE software. Is it one specific protein you are purifying or do you want "all glycoproteins" in the extract? In the latter case I would try using a lot more PVPP and if that does not take care of the phenols use a Q ion exchanger as a capture step.
DTT does not change the protein. One has to be careful though, not too add too much, as it is a much stronger reducing agent than cysteine.
Bo Pontoppidan yes it is a total glycoproteins extract. so you suggest Q ion exchanger?
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