25% is not acceptable for ITC or CD experiments though... I was just sharing our bad experience with a demo nanodrop we had. Even if evaporation is not an issue, one has to take pipetting errors into account when dealing with small volumes. The relative error on 1-2ul is a lot bigger than on 50ul. Unless you want to pre-mix 50ul and use a small quantity of that, which defeats the purpose of miniaturization... It all depends on your applications and sample availability, but if you want a very accurate measurement, miniaturized volumes just won't get you the same accuracy.
Cuvettes will give a better accuracy provided you clean them properly. Just some water or EtOH is *not* enough... Filip Van Petegem On Thu, Jun 16, 2011 at 12:52 PM, aaleshin <aales...@burnham.org> wrote: > I also like our Nanodrop, but I do not recommend using it for Bradford > measurements. > > The 25% accuracy mentioned by Flip is pretty good for biological samples. > Using 50 ul cuvette in a traditional spectrophotometer will not give this > accuracy because cleanness of the cuvette will be a big issue... > > Alex > > On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote: > > I completely disagree with Filip’s assessment. I’ve been using nanodrop > nearly 5 years and never had inconsistency issues. If you work at reasonable > speed (if you put a drop there then lower the lever and click measure before > you do anything else) there will be no issues. At very high concentrations > the accuracy and therefore consistency may become lower. Concentrations > between 5 and 10 mg/ml should be fine. The instrument is pricey though. > > > * Vaheh* > > > > > > > ------------------------------ > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Filip > Van Petegem > *Sent:* Thursday, June 16, 2011 3:34 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good > old Bradford. > > > 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/ > To the extent this electronic communication or any of its attachments > contain information that is not in the public domain, such information is > considered by MedImmune to be confidential and proprietary. This > communication is expected to be read and/or used only by the individual(s) > for whom it is intended. If you have received this electronic communication > in error, please reply to the sender advising of the error in transmission > and delete the original message and any accompanying documents from your > system immediately, without copying, reviewing or otherwise using them for > any purpose. Thank you for your cooperation. > > > -- 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/
