7 replies to this topic

[rick112]

hi

has anyone used NanoDrop UV-Vis spectrophotometers for protein Concentration determination ?
how is the performance of this instrument ??

(have heard that this show lot of variation in measurment )

[mdfenko]

are you using it for reading absorbance at 280nm? if so, there is a lot of protein to protein variation in response.

also, many have found that you should use 2ul, rather than 1 or 1.5ul, for consistent and reproducible readings (regardless of method).

[Zee]

I've read the protein concentration results given by Nanodrop are not reproducible nor accurate due to variability in the types of proteins present in different samples. I tried it once and compared results to concentration values from a cuvette-based spectrophotometer, and the results were inconsistent with no obvious pattern. That being said, I only looked at 2 samples that one time. If someone has gotten the nanodrop to work for measuring protein, that would be awesome!

[labtastic]

I've found nano drops to work quite reliably for protein concentration using 2ul volumes.

However, I have found them to be inconsistent when measuring low concentration samples (OD for 1cm pathlength < 0.3).

Edited by labtastic, 07 July 2013 - 03:31 PM.

[El Crazy Xabi]

It will depend on the chemistry of the proteins you are measuring. Only consistent if you are measuring the same protein.

http://wolfson.huji....cols/od280.html

Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A 280 varies greatly between different proteins; for a 1 mg/mL solution, from 0 up to 4 for some tyrosine-rich wool proteins, although most values are in the range 0.5-1.5 (Kirschenbaum, D. M. (1975) Molar absorptivity and A1%/1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. Anal. Biochem. 68, 465-484).The advantages of this method are that it is simple, and the sample is recoverable.

[Missle]

I use nanodrop for protein readings all the time. It is accurate and reproducible if you have a high purity sample. The accuracy of the instrument is ~ +/- 0.1 so if the A280 of your protein sample is 0.2 then results will have more variability than if it is 1.5. To determine concentration from A280 you need to know the extinction coefficient of your protein. If you know your protein sequence you can use http://web.expasy.org/protparam/ tool to predict the extinction coefficient.

I also use 2ul per reading to eliminate some of the error that can come from technique.

[memari]

There is also another machine:

BioDrop
http://www.biodrop.co.uk/

[Winegarden]

As has been previously stated the results can be variable. Purified proteins vs lysates, and protein to protein variation as you are just using A280. We use a modified micro BCA assay on the Nanodrop that works very well and still conserves sample and we have found this to be more reliable - particularly for lysates.