Arnon Lavie wrote:
~~~
We have been considering buying a Nanodrop machine (small volume, no
dilution needed, fast, easy).
However, while testing our samples using a colleague's machine, we have
gotten readings up to 100% different to our Bradford assay (all fully
purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3
mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how
reliable are the measurements (your thoughts?).
So QUESTION 1: What are people's experience regarding the correlation
between Nanodrop and Bradford?
Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making a 1 g/l solution of gelatin (collagen?)
and see what its A280 is! I noticed recently the
"protparam" tool at http://ca.expasy.org/cgi-bin/protparam
estimates the extinction coefficient given a sequence.
David Briggs wrote:
~~~
I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.
One wonders what does "fine" mean, like same as with Biuret or
Kjeldahl nitrogen, or solution made up by weight?
