That doesn't sound right! ALL of the protein is precipitating? What buffer are you using? What is the DNas/RNase dissolved in - is this affecting pH, organic solvent content, salt concentration somehow? How concentrated is your extract? Do you see precipitation at 37 if you don't add DNase?

The great part of the protein in precipitating, but not all. The buffer contains 50 mM Tris 1 mM EDTA (pH=8) 1 mM MgCl2, 0.14 % DTT and 1x protease inhibitor (roche EDTA free). The lysate is obtained by sonicating the Leishmanial culture (I don't know if this is very important). The solution has no more organic solvents or salts. I don't see precipitation if I don't add DNAse (It is weird!). The approximate concentration is arround 10 micrograms/microlitre. Thank you very much for your time Dr Heath.

Lots of proteins don't like buffers without any salt. So you could try digesting with DNAse in the buffer you described above, but complemented with 100-150 mM NaCl.

Hope this will help

You must have a lot of actin in your extract. DNAse make a complex with actin.

Thank you Yann-Vaï. I wil try to use my buffer complemented with 100-150 mM KCl.

Thank you Dr Sobieszek. I will try to dilute my extract and see if there is a direct correlation with precipitation.

I tried the NaCl but my protein is still precipitating. More ideas? How can I avoid the actin complex formation?

May be you should not worry about the actin/DNAase precipitate. Anyway, what do you want to purify from the lysate?

As said Dr Sobieszek, you probably don't precipitate all the proteins in the lysate. You could see this by SDS-PAGE analysis comparing your lysate, the DNAse (those two are controls) and the precipitated material (simply centrifuge at high speed, discard supernatant, resuspend the pellet in 1X Laemmli). Like this you will see how many proteins are precipitating...

You can also try higher salt concentrations before DNAse (up to 1M NaCl : disrupts ionic interactions), and/or non-denaturing detergents (Triton-X100 or Tween up to 2 % : disrupts hydrophobic interactions), and/or glycerol (improves solubility at low concentration and disrupts hydrophobic interactions at high concentrations)...

Good luck

Thank you very much to all. I will try this points. Dr. Sobieszek, I don't want to purify any protein, I want to perform an IEF with the total proteins from the Leishmanial-parasite lysate to carry out a proteomic analysis.

Hi Christopher, I do not have experience with Leishmanial culture and with the buffer you are using (maybe reported in literature), what I want to share is my experience with human cells. Using both, a classical Ripa buffer or a buffer IEF compatible (8M urea, 40mM tris, 4% CHAPS, 65mM DTT) with DNAse (about 300U/500uL buffer x 20millions cells) I did not have any precipitation problems. Protein concentration was about 1-2mg/mL. Good luck

Hi Christopher, have you tried DNAse digestion with another kind of cell extract? and if you did it in the same conditions, have you seen the same protein precipitation?? because I think that DNAse digestion shouldn't influence protein solubility unless it modifyes some characteristics of the solution. Have you checked the pH of your extract before and after adding DNAse/RNAse? there might be something in your DNAse solution that renders your cell extract acidic and this causes most of the proteins to precipitate. Another explanation is that your DNAse/RNAse solution already has a high salt concentration and this might be causing a "salting-out" effect, which means that solubility of some proteins decrease with high salt concentrations. Good luck with you digestion and keep us informed.

Hi,

High salt cc. should be avoided when the sample is subjevted to IEF. I would use Bensonase (degrades both DNA and RNA in seconds even at low temperature) instead of DNAase.

Do you experience protein precipitation without DNAase after 10 min incubation at 37 C as well? It might be, that the proteins precipitate at 37 C in aither case. Try to keep your sample at low temperature and as I mentioned use Benzonase.

Hi, by 6 days, now, you may have solved the problem. Since the proteomic analysis is the main goal you may add immobilized DNase and after the digestion detach any adsorbed proteins under denaturing conditions

Thank you very much for all the comments. Benzonase was the key. Now it is time to run the 2nd dimension.

For separation of DNA from proteins, in a crude extract I would adjust the salt concentration and pH, load it on a cheap carboximethyl cellulose column, that will bind DNA (positively charged molecules strongly), and collect DNA-free protein solution.

I expect No DNAses - No Protein Precipitate.

The pH for DMA binding should be

One more suggestion - it is not necessary to use DNAse, in order to get rid of DNA. Ultrasonication destroys mostrly such rigud molecules like dsDNA, and even ssDNA. The globular proteins are soft enough for ultrasonication.

Thank you.

Ilya Lyakhov, VARNISS

By serendipity, researchgate has confronted me by this question from 5 years ago... The researcher may have solved his problem or abandoned the procedure altogether... While I am not going to review the lengthy discussion, may I ask if the DNase effect is enzymatic in nature? In one way DNase is releasing histones that are associated with DNA. So basic proteins released may contribute to precipitation of others. In another way DNase itself is a basic protein. You may get rid of DNA by adding protamine sulphate into your sample, if you can check for and exclude any loss of proteins you are looking for /by their associating with the precipitate/.