I have a problem with coloration of a plant protein precipitates.
At the first extraction step i get brown-colored extracts, green - if an object is leafs or stems. And we can't get rid or that coloration even after ethanol or TCA protein precipitation. Protein precipitate still has some brownish coloration, which is stable after gel filtration or centrifugation in Milipore membrane filters.
We are guessing that brown coloration might be caused by phenolic compounds, green certainly caused by chlorophyll.
Maybe someone had the same problem and figured out how to get protein precipitates without coloration? Is it possible that those brown coloring agents could interfere with Coomasie Briliant blue stain and affect the light absorbtion in Bradford assay?
Please, share your experience.
Dear Gabriele,
I think that the browning of your solutions is an enzymatic process between phenolic compounds (namely tannins) and PPO (polyphenol oxidase) enzymes. Additionally, take in mind that tannins can bind irreversibly to and inactivate your proteins.
Your extraction buffer should content protective agents to remove those polyphenols (e.g: 2-5 % w/v PVPP, depending on your tissue) and include antioxidant agents (2-5 mM DTT) to reduce the activity of polyphenol oxidases. Take a look to this protocol. It can be useful.
Best regards.
Oscar
Dear Gabriele,
I think that the browning of your solutions is an enzymatic process between phenolic compounds (namely tannins) and PPO (polyphenol oxidase) enzymes. Additionally, take in mind that tannins can bind irreversibly to and inactivate your proteins.
Your extraction buffer should content protective agents to remove those polyphenols (e.g: 2-5 % w/v PVPP, depending on your tissue) and include antioxidant agents (2-5 mM DTT) to reduce the activity of polyphenol oxidases. Take a look to this protocol. It can be useful.
Best regards.
Oscar
When purifyiing a soluble chloroplast protein, we found that some chlorophyll residue precipitated with the protein when it was precipitated with ammonium sulfate, but was removed at the next step of the purification, which was anion exchange chromatography.
From my experience when using leafs, even in the presence of PVPP and DTT, I got some green crude extracts.
If I want to purify my samples, an Ammonium sulphate fractionation works just fine.
If I want to use the crude, to clean it I apply a syringe filtration step with a low binding protein filter followed by PD-10 Desalting Columns (GE Healthcare).
I hope this helps you.
Regards
Thank you all for your suggestions. I've just tried extracting with DTT and PVPP followed by protein precipitation with alcohol. The results are better. Leaf protein samples contained less intensive colours and root protein samples at low pH buffer was with no coloration at all!
If your aim is to do the proteome analysis (gel free or gel based) of your colored protein sample, you can wash your pellet with 80% acetone containing 0.07 % beta-mercaptoethanol untill your sample become colorless (usually 2-3 washes are sufficient to get rid off the plant pigments). I usually use this procedure and it works well, hope it will work for your samples too.
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