I am trying to purify a plant dehydrogenase, at 65% ammonium sulphate saturation I am retaining all the activity in pellet. But I am loosing all enzyme activity after dialysis. and I am not able to see any bands in SDS-PAGE also. Can any one suggest the solutions for the above problems? As it was unstable at 40c I have 100mM KCl+ 30%glycerol+ 1mM DTT+1mM EDTA+1mM PMSF, pH 9 in my lysis buffer. I have tried 7.5 % to 15% SDS gels. I am getting bands with positive controls . I don't know why no bands are in my sample.
it looks like the protein is getting degraded fast.there are 2 ways to handle this. firstly use a protease inhibitor cocktail protein instead of just PMSF which is only Ser-Thr inhibitor. Secondly Ammonium Sulphate is a bit harsh method, try purifying using a column. If there is no choice, try exploring other ways of precipirating like PEG. Finally, if you know the protein is not so big and made of single chain, try cloning the gene and expressing in heterologus expression system like e.coli
thank you sir, i have tried with protease inhibitor cocktail tablet which had inhibitors for serine, cysteine, and metalloproteases,then also i had lost the activity. as it is a leaves extract have many other interfering substances, so i thought to remove all those with ammonium sulphate.
Can you explain in detail the steps you took between ammonium sulfate precipitation and dialysis? It seems that you lost your protein along the way, since the activity was in the ammonium sulfate pellet but not in the dialysed sample.
By the way, ammonium sulfate precipitation is usually considered to be a suitable early purification step. It generally preserves enzyme activity. The next step could be gel filtration after dissolving the pellet in a small volume. You would not have to do dialysis because the ammonium sulfate will separate from the protein on the gel filtration column.
have you tried to add Polyvynilpyrrolidone (PVP)?
I just watched a defense where the person worked with peroxidase from Phaseolus vulgaris and she obtained very good results by adding PVP (MW 10,000 - PVP-10 from Sigma-Aldrich). She could enhance the enzimatic activity 5-fold in comparison to the crude extract with no addition of PVP (which was pretty low). The same happened after ammonium sulphate precipitation.
PVP will preserve you enzyme from phenolic compounds which are immediatelly released during extraction process of plant tissues. These compounds when are not blocked will impair the enzyme activity by oxidation.
I think it would be worth to try. You should find the optimal concentration of PVP in your extracton buffer that can preserve the maximum activity. In the defense I watched, they started with 10% of the lyophilized crude extract (50 g). If you don't work with lyophilized crude extract, you should relate the PVP amount to g/mL (grams of plant tissue/ milliliters of buffer).
You should also be careful because, when added in high concentration, PVP can inhibit your enzyme activity.
Details of the steps followed: after 60% ammonium sulphate precipitation i have dissolved the pellet in small amount of lysis buffer(100mM Tris containig 100mM KCl+ 30%glycerol+ 1mM DTT+1mM EDTA+1mM PMSF, pH 9), and dialyzed against 10mM Tris containig 100mM KCl+ 10%glycerol+ 1mM DTT+1mM EDTA+1mM PMSF, pH 9 buffer for 15-20h with 7-8 changes.
thank you for suggesting P V P during extraction, i will try to do including that. when i do decolourize my sample with activated charcoal again i loose the activity which suggest that it might in bound form with pigment.
Even if the protein is not bound to the pigments, it might still bind to the charcoal. Can you omit the charcoal step? Ion exchange chromatography might be a good way to separate the protein from the pigments. When purifying CF1 from spinach, we used to get good separation of pigmented material from CF1 protein using anion exchange chromatography. The pigment was strongly retained by the column when the protein was eluted with salt.
even after skipping the charcoal step i am loosing the activity in dialysis, for ion exchange removal of salt is essential rite?. If i do gel filtration, what is the cut off will be good as i don't known the molecular weight of my protein ???
To skip the dialysis by using gel filtration to remove the salt, keep the sample volume as small as possible. Since you don't know the mass of the protein, you may want to try gel filtration media for low and high ranges of molecular weights. Do everything at 4oC and keep fractions on ice.
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