'Fine' for me means comparable to SEC-MALLS measurements and reproducible. I use the E calculated from the sequence using the protparam server at Expasy.
============================ David C. Briggs PhD Father, Structural Biologist and Sceptic ============================ University of Manchester E-mail: david.c.bri...@manchester.ac.uk ============================ http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB ============================ On 16 June 2011 22:31, Edward A. Berry <ber...@upstate.edu> wrote: > 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? > > >