Hi, I have an unknown protein with the following info:
Protein seq:
M S D S E V N Q E A K P E V K P E V K P E T H I N L K V S D G S S E I F F K I K K T T P L R R L M E A F A K R Q G K E M D S L R F L G D G I R I Q A D Q T P E D L D M E D N D I I E A H R E Q I G G
Number of amino acids: 98
MW: 11155.5 Da
Theoretical pI: 4.92
Extinction coefficients: As there are no Trp, Tyr or Cys in the region considered, your protein should not be visible by UV spectrophotometry.
Total number of negatively charged residues (Asp + Glu): 21
Total number of positively charged residues (Arg + Lys): 15
1. I would like to know if there is a difference between using a cation exchange or anion exchange column? and why?
2. If the protein is strongly bound to DNA what column should I use?
3. Is there a way to separate bound (to DNA) and non-bound protein?
Thank you
Based on the acidic theoretical pI, if you do the purification at a neutral pH (7-8), the net charge will be negative, so you should use an anion exchange column (Anion exchange columns have positive charge). (However, that is really just a rule-of-thumb. Cation exchange might work. Even if your protein doesn't bind to the cation exchanger, contaminating proteins might bind, so you could still get some purification just by running the sample over the column and collecting what flows through).
If the protein is bound to DNA, the DNA backbone is also negative, which might help the protein bind to the anion exchange column. Moreover, the DNA will likely obscure several positively charged side chains, increasing the net surface negative charge.
If the DNA is not bound too tightly to the protein, the two may be separated during the salt elution of the ion exchange column.
To separate DNA-bound and DNA-free protein, try a heparin column with a salt elution. DNA binding proteins tend to have some affinity for this resin, so the DNA-free protein may bind more tightly than the DNA-bound protein.
Well, not unknown anymore:
http://www.uniprot.org/uniprot/Q12306
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373614/
Sequence alignment attached in png file (looks like you own G67 mutant).
Your protein has similar properties to src SH3, which I used to work with a couple of years ago, so you can use my old protocol (attached in pdf file, as well as protocol for ubiquitin)
Thank you Adam.
Sebastian, thank you for the info and the purification protocol.
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