Salting Out/Desalting Troubleshooting Qs

[Quantum Buckyball]

Hi All,

I have a question regarding salting out technique using ammonium sulfate.

The pI of my protein of interest is between 4.9-5.3. I noticed there were a lot of DNA and RNA in my protein sample after my initial purification via amylose affinity chromatography. I know that I could use DNase and RNase to get rid of them, but at this point I do not have the funding to get neither of them. I have to come up with alternative ways to approach this.

Hmm, I was wondering if I could use 30% ammonium sulfate to crash out my protein and leave other DNA/RNA behind. Would this work? Would both DNA/RNA co-crash out with m protein of interest?

My protein is currently in a buffer containing 10 mM of maltose and is at pH 7.6. My PI recommended using desalting column to separate them so I should also give that a try.

[aberrant]

Did you confirm that "there were a lot of DNA and RNA" by measuring your elution/elutant's 260:280 ratio?

 

by desalting, do you mean a gel filtration/size exclusion? that can only separate the non-binding DNA/RNA

 

if you are trying to break that electrostatic interaction, you can give ion exchange chromatography a shot.

[Quantum Buckyball]

Yeah, the ration is about 1.3~1.8 .... I have tried DEAE column with a gradient method 0 through 100 % of 500 mM NaCl. The result from DEAE was not good, it appeared that my protein did not bind well with it.

[Quantum Buckyball]

Hi,

Thank you for your reply. I have figured out a way to solve my problem today and the result was terrific!

[aberrant]

How did you solve your problem? A negatively charged column? (DEAE = positive charge, hence it probably doesn't bind well with DNA/RNA-binding proteins)

Edited June 16, 2013 by aberrant

[Quantum Buckyball]

How did you solve your problem? A negatively charged column? (DEAE = positive charge, hence it probably doesn't bind well with DNA/RNA-binding proteins)

I borrowed some DNase and RNase from another lab lol.

[ion_exchanger]

I borrowed some DNase and RNase from another lab lol.

 

Aww!! That was the first thing that I thought of when I first read this, because that's what I would have done, and that's what the lab next door often does! I was thinking about this question for a while. Nice to see you solved your problem. Pesky proteins.

[Quantum Buckyball]

How did you solve your problem? A negatively charged column? (DEAE = positive charge, hence it probably doesn't bind well with DNA/RNA-binding proteins)

That is true, typically both DNA and RNA would get eluted off DEAE column first, and then your protein would get eluted out later on after you increase the concentration/strength of salt. However, it was not the case for my protein. My protein got eluted out around elution fraction 20.. and then bunch of DNA/RNA got eluted our around elution fractions 40~70. I was like WTF? lol

[Quantum Buckyball]

Aww!! That was the first thing that I thought of when I first read this, because that's what I would have done, and that's what the lab next door often does! I was thinking about this question for a while. Nice to see you solved your problem. Pesky proteins.

Yeah I'm glad I got it to work eventually, it was a busy day for me in the lab. I tried 3 different purification techniques and finally got 1 to work lol