how to confirm the presence or absence of very small dna (if below 5 bps or bases) in a purified protein sample

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Tue Feb 23 17:10:37 EST 2010


Dear Uday,

Actually, the A280/A260 ration depends on the individual AA
composition of your protein.

5 bp DNA should be roughly 1 kDa in size, so it might be removable by
dialysis or ultrafiltration as log as it does not bind too strong.

If you suspect your protein binds specifically small junks of DNA, the
situation is more difficult. If it is non-specific binding, you might
be able to demonstrate the effect by affinity chromatography on
immobilized DNA or oligos.

If you think that your protein has bound some DNA, you maybe can
detect it by fluorescence: Either autofluorescence of DNA or binding
(and or change of fluorescence properties) of dyes like
ethidiumbromide, propidiumiodide, H33258, DAPI, SybrGreen and so on.

If you know the identity of your protein (i.e. if you can get hold of
the AA sequence) life will be easier.

In order to determine the sequence of the bound DNA, try to clone and
sequence it.

If you just need to make sure that your protein preparation does not
contain any DNA, try an appropriate DNAse.

HTH

Wo


More information about the Methods mailing list