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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 <[log in to unmask]>:
> 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 <[log in to unmask]> 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:[log in to unmask]] On Behalf
>>> Of Filip Van Petegem
>>> Sent: Thursday, June 16, 2011 3:34 PM
>>> To: [log in to unmask]
>>> 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 <[log in to unmask]> 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: [log in to unmask]
>>> 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: [log in to unmask]
>>> 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: [log in to unmask]
>> http://crg.ubc.ca/VanPetegem/
>
>
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