I have used the nanodrop and like it, although I have also seen some variation, even sometimes a concentrating trend over ~30sec due to evaporation I think. So, I always spot the protein then measure instantly, and it is relatively consistent. But overall the convenience is excellent, and worth the modest price. It really was a very nice invention!
JPK On Thu, Jun 16, 2011 at 4:33 PM, Filip Van Petegem <filip.vanpete...@gmail.com> wrote: > 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/ > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *******************************************
