I am trying to purify my protein (protein implicated in the DNA recombination process) which was overexpressed in benthamiana plants using the FPLC system, so, I grind my infiltrated leaves and extract the total proteins using the buffer: Tris 100mM Tris ph 8.0, 5mM EGTA, 5mM EDTA, 150mM NaCl, 10mM DTT.
After centrifugation at 20,000g 2 times (to clarify the supernatant) I notice that my precipitate is gelatin like (could be DNA). A western-blot test of the precipitate showed my protein is in the majority of the precipitate.
- Could anyone suggest a method to decrease the viscosity of my protein extract, keeping the complex protein-DNA intact?
- How can I show, by a quick experiment, the interaction of my protein with DNA (protein is tagged) and have an idea about the nature of DNA interacting with my protein?
To reduce DNA associated viscosity, you can use sonication to fragment DNA, or DNAse (MNase for example) treatment (your protein will still be associated to DNA as there will be a "footprinting", protection of the DNA associated to your protein against the DNAse activity). You can look at the first steps in protocols for ChromatinIP that should give you some informations about such treatments.
If your protein is ossociated to DNA, during/after purification you can monitor the absorbance at 280nm(protein) vs 260nm (DNA). A strong 260nm absorbance will assess the presence of DNA together with your protein.
You could also imagine to perform some gel shift assays between your protein alone (after a high salt wash for example) and your protein associated with DNA
To determine the nature of DNA associated to your protein is more complicated, the only way I see to have a real identification is a CHIPseq like method.
I agree with Nicolas Lévy answers.
To reduce your viscosity, an other way is to change your purification system, using antibodies against your protein. For example using IP protocols. This way, you will have only your target protein and the associated DNA.
With purified protein, the work will be easier to identiy anything.
**** to decrease your viscosity you could try to sonicate your cells in your "protein extraction buffer" at the beginning.
**** you can detect the presence of DNA using UV 260nm (in some FPLC you could directly have 2 wavelengths in same time), and you compare to the UV 280nm showing your protein
I agree with Nicolas Lévy. you can also use a few random restriction enzymes to sheer the DNA in your sample. this will leave large chunks of DNA in the sample, but will decrease the viscosity.
viscosity depends on DNA/RNA content therefore the best way is to decrease the concentration of DNA/RNA by using enzymes as mentioned above, and after the 1st step of purification can be IEX with large size of beads/media because DNA is typically has a negative charge.
I think probably you can try 1) after grinding the leaves and extracted with solvent, do a quick run to remove any debris with lower centrifuge force instead of at 20000, so most of your protein and DNA are still in supernatant;
2) Then run gel-filtration column to separate DNA and protein-DNA complex.
3) precipitate your protein-DNA.
In addition, the gel-like stuff may not be necessarily DNA, it could be polysacharide, or large proteins as well.
4) Could you try protein precipitation with salt ((NH4)2SO4), or even Organic solvent if your are not worrying about the structure
At high viscosity it will be hard to run any type of column chromatography by using just gravity force, option is to use pressure with a pump but it might take a lot of time to optimize it, therefore the 1st step should be decreasing the viscosity by using any method suggested above.
Thanks a lot for taking the time to answer my question. All of these answers are helpful for me, I really appreciate it. Thanks all
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