DNA carry-through in protein purification is quite common, especially if
you are working with basic proteins. A Talon step certainly doesn't
guarantee the removal of nucleic acid, and large fragments will
certainly run in the void volume of your gel-filtration column.
You can try washing the Talon column in High Salt buffer once your
protein is immobilised to remove any nucleic acid - i.e. a buffer
containing 750 to 1 M NaCl. Wash it back to the salt you want and elute
as before.
DId you include any steps in your protein purification to remove nucleic
acid, i.e. including DNase or Benzonase in your lysis buffer? Have you
tried Protamine Sulphate precipitation?
Measure a UV spectra of your protein, post gel-filtration - have you got
large maximal absorbance at 260nm - rather that at 274-280nm? This is
probably the best indicator of DNA/RNA contamination there is.
Run some of your protein on either an agarose or native acrylamide gel.
Stain with ethidium bromide or similar. Can you see anything?
Hope this answers some of your questions.
Regards,
Antony.
--
Dr Antony W Oliver
Cancer Research UK: DNA Repair Enzymes Group
Section of Structural Biology
The Institute of Cancer Research
237 Fulham Road
LONDON SW3 6JB
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020 7153 5488
--
The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.
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