I would add that there are some issues with air, you have to be careful with nanodrop that the path is ok, and also if concentrations are low, <1 mg/ml for instance, i am not sure one can trust it - compare 50 ul 1 cm path results with nano at 0.5-1 mg/ml... i get inconsistency there.. its good for concentrated samples and fast. and handy, we use it a lot, but for dilute samples, i always use 1 cm light path. just my feel on it.
Tommi On Jun 16, 2011, at 11:20 PM, aaleshin wrote: > Filip, > 25% accuracy is observed only for very diluted (OD280< 0.1) or concentrated > samples. But those sample a rarely used for ITC or CD. The concentrated > samples require dilution but a regular spec does it too. Since the light > passway is very short in Nanodrop it is accurate with more concentrated > samples, which we crystallographers use, so Nanodrop is ideal instrument for > our trade. > > If the drop is within recommended volume like 1-2 ul for our model, its size > has a very small influence on the measurement. > >> Cuvettes will give a better accuracy provided you clean them properly. > I hated those times when I had to measure a concentration because of a need > to wash a cuvette. In a biological lab they are always dirty. We switched to > plastic disposable cuvettes for that reason... > > Alex > > On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote: > >> 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/ >
