Dear Arnon, I have a Nanodrop2000, which reads from the post or a user supplied cuvette. I have had NO complaints about using the Nanodrop for reading protein concentration immediately prior to crystallization setup. When I have observed differences in OD280 vs Bradford, it is usually due to one of the following:
1) The protein is deficient in the amino acid residues that provide the 280nm signal. This can be corrected with the extinction coefficient, and can be programmed into the Nanodrop so that the readout is correct. 2) The protein is behaving badly in the Bradford assay (interference with some component of the protein buffer) 3) The dilution used in the Bradford is contributing to large concentration errors (and can be combined with 2, above) The value of the Nanodrop, is that you get a OD280 that you can reproduce prior to every experiment at a low cost of protein sample. The N2000 can also do the Bradford or other assay on the post, or with a cuvette if you really want to do it that way. When there have been no mitigating factors, my Nanodrop OD280 readings have been within 5% of the values I get from SEC-MALS, MassSpec or Bradford. I have no experience with the Pearl, but I am very happy with my Nanodrop 2000. Good luck with your choice! Bryan -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Arnon Lavie Sent: Thursday, June 16, 2011 3:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford. 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/ ***********************************************************
