Dear Arnon, the Bradford method is not recommended for accurate measurements. The readings are strongly dependent on the amino acid composition. A much better method is using the absorption at 280nm under denaturing conditions (6M Guanidine), and using calculated extinction coefficients based on the composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds). This method is also old (Edelhoch, 1967), but very reliable.
One thing about the nanodrop: smaller volume = more evaporation. On the demo we've had, I was so unimpressed with the precision (>25% variability between two consecutive measurement) that we didn't consider this instrument at all. So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But most respectable spectrophotometers will take cuvettes with 50ul volumes - a big step up from 1ml volumes... Filip Van Petegem On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie <la...@uic.edu> wrote: > Dear fellow crystallographers - a question about spectrophotometers for > protein concentration determination. > > We are so last millennium - using Bradford reagent/ 1 ml cuvette for > protein conc. determination. > > 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? > > While researching the Nanodrop machine, I heard about the Implen > NanoPhotmeter Pearl. > So Question 2: Is the Pearl better/worse/same as the Nanodrop for our > purpose? > > Thank you for helping us to advance to the next millennium, even if it is > nearly a dozen years late. > > Arnon > > -- > *********************************************************** > Arnon Lavie, Professor > Dept. of Biochemistry and Molecular Genetics > University of Illinois at Chicago > 900 S. Ashland Ave. > Molecular Biology Research Building, Room 1108 (M/C 669) > Chicago, IL 60607 > U.S.A. > Tel: (312) 355-5029 > Fax: (312) 355-4535 > E-mail: la...@uic.edu > http://www.uic.edu/labs/lavie/ > *********************************************************** > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
