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Hi,
Typically one does not expect massive
contamination with DNA unless the protein you work with is either very basic,
or is a DNA/nucleotide binding protein. Having said this, I must add that in
almost every IMAC preparation that I have *ever*
run, regardless of the resin used, there was always a small but detectable
amount of nucleic acid together with the protein. This becomes very evident in
the second step which often is done by ion exchange using some form of a
quaternary-amine resin. The nucleic acid separates very nicely and elutes as a
separate peak at around 35 mS/cm (where very few proteins still stick to the
resin) with 260/280 ratio > 1.50. What you have, assuming that the third
peak is predominantly protein, is kind of like that – the first peak is
probably DNA aggregates (cross-stitched by various ‘sticky’
proteins). Not sure about the second peak – I don’t know what
column was used and where its void volume might be, etc.
So, in conclusion – DNA contamination
of IMAC preps is real and common, the amounts vary – if you have abundant
protein of interest and you’re using a correct ratio of protein to resin,
the amount of DNA would be less, however if you’re using a large excess
of resin to protein (and/or your protein is just isn’t abundant), the DNA
amounts can be comparable to that of protein. As you mention, it does not take
much DNA to overpower the protein signal, since the specific absorption of DNA
is usually much higher than that of (most) proteins.
Two easy methods seem to minimize DNA
contamination of ‘regular’ protein preps – a) washing the
column with 1M NaCl prior to elution or b) using ion-exchange as the second
step. In special cases it may be very hard to resolve the DNA away, and in some
hellish cases (like one of the proteins I am working on right now) the removal
of DNA resuts in irreversible precipitation of the protein of interest.
To detect DNA you could try staining with
SYBR green or something like that.
Good luck,
Artem
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of Jacob Keller
Sent: Tuesday, January 08, 2008
4:20 PM
To: [log in to unmask]
Subject: [ccp4bb] DNA
contamination post-Talon?
Sorry for the off-topic question again,
but has anybody encountered DNA fragments' sticking to a Talon column?
I got the attached trace after putting my Talon-purified protein onto an s75
column. Notice that the A254:A280 ratio changes pretty dramatically from the
first peak to the last (although the scales are off a bit--the red trace should
be about half what it is. This actually happened quite a while ago, so there
are some experimental gaps (such as a coomassie gel of all fractions), but from
the post-Talon gel, the protein looks about, say, 90% pure (probably
better)? I am trying to make sense of this in light of some more recent
developments.
Could it be a contaminating protein bound to nucleotide or some other
highly-absorptive small molecule in the void volume? I would not think
that would have enough absorbance, really, to be seen so
prominently. Or perhaps fragments of DNA, which have a much greater E254,
and might run in the void volume if sufficiently large, but I would not think
they would make it past the Talon (hence the question)...Any suggestions
welcome...Also, from the dried coomassie gel, could I determine whether there
is DNA there? How can one detect DNA in PAGE gels?
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
lab: 847.491.2438
cel: 773.608.9185
email: [log in to unmask]
*******************************************