Hello Justin and others, The volume comment I make is based on mixing prior to the experiment, e.g. with Bradford reagent, Guanidine for the Edelhoch method, etc.
Any direct measurement of A280 of protein samples requires you to know the extinction coefficient, which depends on the amount of Tyrosines and Tryptophans in the protein, but *also* the folding, as shown almost 5 decades ago. One way of minimizing this error is denaturing your protein with e.g. 6M Guanidine, at which point the extinction coefficient becomes largely independent of the protein composition, resulting in a more accurate concentration determination.( http://www.chem.uky.edu/courses/che554/Photometry/Edelhoch1967.pdf ) Bottom line: if you just put your protein on the nanodrop, and rely on the A280 absorption, there will be a systematic error. If you want to use e.g. Edelhoch's method, or any other method that requires mixing, the error will be larger for smaller volumes. cheers Filip Van Petegem On Thu, Jun 16, 2011 at 2:08 PM, Justin Hall <hallj...@onid.orst.edu> wrote: > Hi Alex, > > I read Filip's comment about volume not as a path length argument, but > about concentration uncertainty in mixing small volumes to dilute a sample > down before measuring it (?). I have never had to make a dilution for my > nanodrop (my proteins are usually not that concentrated), but I could see > his point if I did have to. > > As for the variance between samples, I don't know about >25%, but I have > observed multiple readings to have variance. I always take 3 readings on my > nanodrop and then average them to deal with the variance I see. I don't mind > doing this because the instrument is so fast, and I don't mind the cost at 6 > ul of sample total. > > The most variance I have seen is usually in spin columns, where I will be > doing a buffer exchange from a storage buffer (sometimes at ca. 20% > glycerol) into an assay or xstal buffer, and I have wondered to myself if > the variance I see could be due to incomplete mixing of a protein sample > betwen a viscous buffer at the bottom with the rest of the buffer. I don't > know how often other people find themselves in a situation where they may be > sampling their 2 ul from a "micro-environment" that is not homogenous with > the rest of the sample, but with small volumes I think that be a problem. > Food for thought. > > Filip, I would buy a nanodrop. It is much better than a Bradford/cuvette > and your students will love you for it. Cheers~ > > ~Justin > > > > Quoting aaleshin <aales...@burnham.org>: > > 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/ >>> >> >> >> -- 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/
