I basically agree with Stefan Gajewski and David Horita: either run your protein over a heparin column or an anion exchange column. Heparin mimics the DNA backbone and will thus displace the DNA from the protein. Considering your low pI, it might not be advisable to perform anion exchange chromatography step. If your protein is negatively charged (which will happen at a pH more than 6), both the protein and the DNA will bind to the column.

Just employing a DNase such as Benzonase might help if DNA is not bound that tightly, but in many cases this treatment will not be suffient since the protein will protect the bound DNA from being incised.

For purifiying DNA/RNA binding proteins, I would suggest to do the following: you add some DNase already into the cell lysis buffer (this also makes your cell lysate less viscous). As an optional step after cell lysis you can add LiCl at a final concentration of 1M to precipitate the DNA. A centrifugation step (more than 25,000g) will clear the lysate from any insoluble molecules, such as the precipitated DNA, cell wall fragements and protein aggregates. In the optimal case your protein carries an affinity tag which allows you to perform a first affinity capture step. Then you purify the protein with the heparin column. Afterwards you can do a polishing step employing size excusion chromatography. This procedure should give you a pure, DNA-free and homogeneous protein sample!

you can try to include DNase in your lysis buffer. DNase I works best if your salt concentration in the lysis buffer is low (

I usually use this to get rid of DNA bound to proteins :

- Use DNAse or benzonase in the lysis buffer to cleave DNA (avoids copurification of DNA-binding proteins due to large DNA molecules, and lowers viscosity of the sample).

- Bind protein on IMAC (Buffer + 20 mM imidazole and 500 mM NaCl...)

- Wash with lysis buffer.

- Wash with a high ionic strength buffer without imidazole (1 M NaCl is fine) : DNA is eluted and protein remains bound to Nickel.

- Wash again with lysis buffer to lower salt concentration.

- Elute your protein.

Have you tried benzonase? This is a very usual nuclease that is part of the downstream processesin high quality assurance labs, including those producing vaccines. It is available at several distributers inlcuding Sigma-Aldrich and Millipore.

I think this is a common problem.

Try...

Benzonase/DNase in lysis

After initial affinity try heparin column to polish.

Ion exchange and hydrophobic columns are also useful. Monitor 260 and 280 during elution (collect correct fractions) requires dual wavelength

I've been told pei can precipt Nucleic acids over protein but in my experience will precipt both. (Worth a try. )

Failing that... 8m urea and re-fold after removing Nucleic acid by ni column.

Most lysis methods cause the release of nucleic acids (DNA and RNA). These have to be removed because they can cause viscosity problems or due to their interference with subsequent chromatographic steps. Different methods exist:

nuclease treatment does not necessarily remove bound DNA although it is likely to remove exposed or free DNA. High salt buffers should disrupt DNA binding of the traget protein. This will obviously require some individual testing, but many proteins won´t bind DNA in vitro at NaCl or KCl conentrations above 400 mM (there are exceptions though that will still bind DNA at 500-800 mM) but it is anyway worth a shot. High salt does not interfere with many resins, including Ni-NTA.

Think vice versa. Try to purify DNA, not proteins. Like it happens during extraction of DNA for PCR. Add Guanidine Thiocyanate, then silicagel, then washing buffer... Instead of discarding the supernatant during the purification of DNA use the supernatant to purify your proteins on Ni column. Luck.

Hello,

I have seen that many people recommand Benzonase. It works, BUT keep in mind that if you do your purification at 4°C Benzonase is not as effective as at 37°C.

You can try to add the couple 30 mM Spermin/50 mM DTT in you clear lysate to precipitate DNA. Then just centrifugate and check if your protein is still in the supernatant.

Depending on the properties of your protein, you may be able to get rid of DNA using hydroxyapatite resin.

As commented, DNAase in the lysis buffer should take care of the problem. You might get short bits of DNA bound to the binding site, but that should not be a problem.

First, are you trying to retain DNA binding activity of your protein? If you are, do not use DNAase, nucleases or denaturants such as guanidine. If you add a nuclease and plan to do anything with DNA later, you will have a nuclease contamination problem. Depending on how your protein, ammonium sulfate PEI can be used as a first step. We use ion exchange prior to nickel chromatography. Then during nickel chromatography we use high salt washes (depending on how your protein handles salt) of up to 1.5 - 2 M NaCl. We also use hydroxyapatite as suggested above. Using a combination of these we never have any DNA in our protein preparations.

I typically use sonication to for lysis with protease inhibitors in the lysis buffer and then allow the lysed celles to rock at room temperature for 30 minutes to an hour with lysozyme and benzonanse. This has always worked well for me to yield uncontaminated protein.

We add every time Benzonase to our digestion mixture for instance, since this compound is great to reduce heavily the presence of DNA/RNA , thus keeping the solution not viscous at all. As Alexander said, depends on your reaction condition: Merck did a great quality control of the Benzonase, which I suggest you to check for optimal pH, temperature etc in which the compound works well.

What do you know about the DNA-binding properties of your protein? We routinely use Benzonase (30-60 minutes following lysis) and high salt washes (0.5-1.0M NaCl) to remove nucleic acids from high-pI proteins, but here we're not trying to disrupt a high-affinity, specific interaction, just a non-specific electrostatic attraction. If, e.g., your protein binds with nM affinity to a tetramer, then Benzonase might not be enough. On the other hand, if it is known that high phosphate disrupts binding, then that would be something to add to your wash buffer. Sometimes ion exchange will really help, either before (as Michael Sehorn suggests) or after Ni-NTA. DNA will bind to a Q-type column (I would advise against loading lysate on a Q, but that's another issue), and some DNA-binding proteins will stick to an SP. Even if the overall pI of your protein is 6, a large patch of Arg/Lys residues can be enough to get your protein to stick a bit. Also, for DNA-binding proteins, phosphocellulose sort of acts both as IEX and affinity resin and might be able to compete away your DNA.

If your DNA contamination is significant and persistent, try Polyethyleneimine precipitation followed by AmSO4 precipitation as initial capturing step.

Everybody hates this protocol, but it usually works quite fine.

You can add Heparin Sepharose, DNA cellulose, Hydroxypatite or Phosphocellulose chromatography after the affinity step. Those matrices bind protein similar to (or exactly like) DNA so you expect only DNA free protein coming in the peak fraction.

Phosphocellulose is a cheap low capacity media, DNA cellulose is not very stable, Heparin Sepharose is expensive but else excellent and Hydroxypatite needs sometimes a lot of optimization to resolve proteins well but is else a very good matrix.

Also a good idea is to use ~50mM Phosphate buffer for lysis. I found that quite helpful for DNA binding proteins.

I am currently working with an DNA binding protein purified by Ni column. As suggested by Michael Sehorn and David Horita, I used 1M NaCl in my lysis buffer and wash buffer(usually 300mM in Ni-NTA buffer). There was nearly no DNA contamination in by protein sample. If 1M NaCl does not work quite well, I suggest you can use DNase to treat the sample before and after the Ni-NTA chromatography.

50mM EDTA added to your sample, just if you have long lunchbreake to run additional Size-Exclusion equilibrated with the buffer EDTA-free. This is suggestion only of the old lazy guy likes to start with the simplest method at the beggining;)

Proteins are removed through extraction of the aqueous phase with the organic mixture of phenol and chloroform or soluble proteins are separated through mixing with chloroform and centrifugation.

Dear Jiahui

I had similar problem in tissue 2DE with DNA contents of my samples.

You can use one of the following commnets:

1. Percipitate protein with acetone or TCA/acetone

2. Extract DNA from your samples by phenol/chloroform method.

3. Use DNase to destroy the DNA content of your samples.

I use this type of DNase:

I basically agree with Stefan Gajewski and David Horita: either run your protein over a heparin column or an anion exchange column. Heparin mimics the DNA backbone and will thus displace the DNA from the protein. Considering your low pI, it might not be advisable to perform anion exchange chromatography step. If your protein is negatively charged (which will happen at a pH more than 6), both the protein and the DNA will bind to the column.

Just employing a DNase such as Benzonase might help if DNA is not bound that tightly, but in many cases this treatment will not be suffient since the protein will protect the bound DNA from being incised.

For purifiying DNA/RNA binding proteins, I would suggest to do the following: you add some DNase already into the cell lysis buffer (this also makes your cell lysate less viscous). As an optional step after cell lysis you can add LiCl at a final concentration of 1M to precipitate the DNA. A centrifugation step (more than 25,000g) will clear the lysate from any insoluble molecules, such as the precipitated DNA, cell wall fragements and protein aggregates. In the optimal case your protein carries an affinity tag which allows you to perform a first affinity capture step. Then you purify the protein with the heparin column. Afterwards you can do a polishing step employing size excusion chromatography. This procedure should give you a pure, DNA-free and homogeneous protein sample!

Removal of Proteins is deproteinization.....Most common method for removing proteins explore the differences between proteins and nucleic acids in organic solvents.the differences include difference in solubility ,difference in partial specific volume etc.

Nucleic acids are hydrophilic molecules and proteins contain many hydrophobic residues making them partially soluble in organic solvents.

the organic solvent used are phenol and chloroform containing 1 % isoamyl alcohal( Kirby And Marmur Method)

First having a cell lysate you can either change a buffer composition (for a pilot experiment clever people use 96 well plate) salt, pH, ionoc strength, depends what do you know about DNA binding of this protein, or just use benzonaze. In the last case do remember to wash the ninta or Roche Ni (EDTA resistant) column very very well befor you elute this protein.

3M Nacl will do the trick.

Treating your samples with DNases/ RNases/Benzonases can be pretty costly. Alternatively, you can remove your nucleic acids by precipitation with Protamine sulphate.

Benzonase worked best with us.

Indeed! Benzonase has been reported to be free of detectable proteolytic activity and it is very specific. Normally it works in a broad range of experiment conditions, but I suggest to have a look at this https://www.millipore.com/publications.nsf/a73664f9f981af8c852569b9005b4eee/86011a277ec4ff9b85257a02005c7a48/$FILE/72209_emd.pdf

for buffer and pH compatibility.

Hope it helps,

Stella

Two possible routes here

1. separate on a hydroxyapatite column running in 2M Nacl 5M urea 20mm phosphate. DNA and RNA bind to HAP proteins pass through

2. Separate by ultra centrifugation by isopycnic centrifugation on CsCl or CsSO4 gradients containing GuHCl or urea

If you want to release DNA, you can use anion exchange chromatography. DNA will bind to the resin more tightly than the protein. Depending on the protein you may use a flow through method or bind and elute. I'd try DEAE or TMAE and pH ~7 if your protein is stable there.

Like others have suggested, heparin chromatography can work well for this. However, I have found that the best way to remove co-purifying DNA and associated DNA-binding proteins is to perform size-exclusion chromatography (S200) under high salt (1-2 Molar) conditions. Analyze fractions by UV spectrophotometry and SDS-PAGE. You should see DNA come out in the excluded volume (high absorbance, no bands on gel), and proteins elute at their monomeric MW, unless they form tight oligomers. You will likely have to concentrate your sample and exchange the buffer prior to further analysis.

I would avoid nuclease treatments if you plan to study protein-DNA interactions after purification.

Treating your samples with DNases or Benzonases can be  costly. Alternatively, you can remove your nucleic acids by precipitation with streptomycin sulphate or protamine sulphate or lithium choloride or 3M NaCl.

We routinely use a very active nuclease with no problems. Please see here: http://www.protean.cz/en/recombinant-protein/11/endonuclease-s-marcescens

This discussion is so funny! Really.

If anyone wants to separate DNA-binding protein from non-DNA binding ones in order to get it purified, the separation is done by dissolving the low-purified protein-DNA complexes in higher concentration of Tris-HCl/NaCl buffer (like 20mM Tris/50mM NaCl) heated moderately for separation of the DNA (at 55-65oC) rapidly cooled and immediately immunoprecipitated with carrier-bound (like Sepharose) anti-human- (if the protein is from human cells) -double-strand DNA-antibody. This method at this stage leaves bound the DNA-protein complexes. A detailed procedure is described here: 

http://cancerres.aacrjournals.org/content/60/14/3921.full#B17#B17

Another good method for entire purification of DNA-binding proteins is given in a "classical" paper here:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC334001/pdf/nar00225-0017.pdf

Thank you I found this helpful too.

If you want active DNA binding protein, I would elect to choose a different choice than proposed by Mr. Funny.  

Michael,

How would you recomend it to be done? Other than what you posted since I don't have the budget for the chromatography. At least that's what they say! I don't think messing up my experiment being funny. Really. 

Micah

I know I am 6 years late to the party and you already completed your project and submitted your publication. But high salt wash (500 mM NaCl) disturbs the binding of DNA to protein because the binding is mainly electrostatic.

high salt(1-2M) with extensively washing step from affinity column normally works well

anybody can suggest me, how to remove the nucleic acid (DNA/RNA) from purified human immunoglobulin (ivIg)????

Micrococcal nuclease is highly efficient, and it can be inactivated by heating at 65 C for 10-15 min. However, make sure that your protein refolds properly by cooling the reaction mix at room temperature, and then place on ice. I used this method for few experiments in the past.

Maybe you can try Streptomycin?