well, actually you can use the bradford reagent with a nanodrop... we have one in our lab and I did it once. the results I got from the bradford were comparable to those I got with a 280 absorbance of my purified protein (the 280 nm reading is only suitable for purified proteins, I guess) AND with a common bradford method with cuvettes... I think it depends on the model you have, maybe not all the nanodrop versions can and have this option. Maybe the dynamic range is somewhat narrower than with cuvettes, but I should check... The procedure is a bit long, don't you have a manual of instructions with your machine? I can send you a scan of mine, maybe...

I use the Bradford reagent from Bio-Rad, diluted 5-fold with water, and make the measurements in a 96-well plate format, 200 µl sample volume (including Bradford rgt.), absorbance at 595 nm. With BSA as a standard, the assay can readily detect 1 µg of protein.

Hi Mark.

In my research projects, I often use NanoDrop A280 measurements for estimating soluble protein concentrations. The method is fast, cheap, and only requires few µl of sample. However, you are correct that when conducting consecutive measurements, it is important to wash the detector to avoid residual protein interfering. I always conduct a few blank measurements in between to ensure no protein “carryover” is present, and it does not seem to be a problem with washing. As long as the NanoDrop system is well maintained, in my experience the repeatability is good!

The protein concentration estimations are, however, usually off from Bradford or BCA measurements by a factor of two or more! Therefore, I would only use the method when you do not require a very accurate protein concentration estimation.

A few things you should consider, which may affect the accuracy of your measurements:

1. NanoDrop A280 measures absorbance of mainly tryptophan and phenylalanine, and makes estimations to the average frequency of these amino acids in your protein mixture. Therefore, you may get different estimations when measuring complex protein mixtures and purified proteins.

2. NanoDrop A280 is in my experience very sensitive to interference from a number of compounds, including surfactants. Therefore, be sure to use a good solution for the reference measurements.

3. Consider doing dilution series, and dilute with an appropriate solution which takes point 2 into consideration.

I usually estimate the protein concentration with Bradford or BCA, and adjust the NanoDrop A280 measurements accordingly when this gives meaningful data.

Hope this answers at least some of your question, and good luck with your research!

/Tue

Thanks for the tips, Tue!

So just to finalize what you have said about "estimating the protein concentration with Bradford or BCA" - you do it also using the nanodrop machine? Bradford/BCA I mean.. (oh yes, the model we have is ND-1000.. is there any other model?!)

Hi Mark,

No problem - happy to help!

I suppose it would be possible to use the ND-1000 for BCA/Bradford (I am very fond of this model myself). However, I use 96 well plates for BCA/bradford and measure the absorbance with a Tecan plate reader. This is motivated by the fact that I always analyze a dilution series in technical triplicates my sample (as I only use the assay for when I need accurate measurements). It, therefore, amounts to a lot of measurements on a ND-system which takes time, and the reactions in the assays are time dependent. Therefore, you will likely get a drift in the measurements, but I do not know how severe.

Hi Tue,

I see. What I need now is actually to know a total lysate protein concentration (to load appropriate amounts on a gel for WB). So I think what I need now is just a couple of measurments and, according to your words, ND (@A280) is fine for that...

Right?

Hi Mark,

For normalization of total protein material from a total lysate for WB gel, I would think that a simple ND A280 would be just fine, of course depending on your experimental setup.

Any offset in ND A280 total protein concentration estimation would be the same across all gel-lanes if the sample material is similar (in my experience the ND A280-system has a high precision). An example would be that you estimated your gel-lane load to 20 µg with ND A280, but you actually only loaded 15 µg total protein. As this is true for all lanes, you will still be able to compare across the different lanes. This should be just fine if you e.g. have two similar cell lines/sample lysates and are using WB to measure expression of a given protein in the one. However, if you are having a complex experimental setup with dilution series, etc ND might not be accurate enough, so it really depends on your experiment.

Hope this makes sense, else feel free to ask again!

 Hi dr Tue

I understood that no need to use a kit for estimating protein concentration when we use nanodrop..am I correct?.. if yes, is there any paper mentioned this method?

Hello,

Caution when measuring proteins on the Nanodrop in small volume. Proteins often lack surface tension which is necessary for measuring with the Nanodrop as it uses the surface tension of the sample in order to stretch it into a liquid column. Prepare to frequently recondition the pedestal if you decide to use the Nanodrop for protein measurements.

Hi Alisha Simmons could you please elaborate on what you mean by "frequently recondition" the pedestal? thanks a lot!

Hi Bhavana Nayer,

If the cohesion of the molecules in the sample is not strong enough, the measurement column breaks and the readings will show inconsistent and inaccurate values. Many samples utilized in the lab lack surface tension e.g. most proteins, organic solvents, detergent buffers and alcohols.

Therefore, the pedestal needs to be reconditioned on a regular basis to maintain the hydrophobic properties of the surface area. The ND pedestal is made of stainless steel which is not inert so sample residue starts building up. NanoDrop recommends reconditioning of the pedestal every six months in order to remove this build up, which adds costs and requires time for the procedure. Here is the link to the instructions for reconditioning: http://tools.thermofisher.com/content/sfs/brochures/T005-NanoDrop%201000-&-NanoDrop%208000-Cleaning-and-Reconditioning.pdf

I would suggest observing your measurements carefully as the pedestal might become unconditioned in a shorter time span depending on the samples you apply. Hope this helps!

Thank you Alisha Simmons ! That was very helpful. I will do that on a regular basis from now onwards.

I have been extracting protein from shellfish mantle tissue using a Trizol protrocol and then dissolving my final pellet in a solution of 8M urea with 0.1% SDS. I have noticed that when using ND A280 my readings are inconsistent...could this be due to the detergents I am using?

Please see this document for troubleshooting information:

https://biosci-batzerlab.biology.lsu.edu/Genomics/documentation/Nanodrop_Protein_Guide.pdf

Fisher website says the Nanodrop is fine with purified proteins, but they don't recommend using it with complex mixtures of macromolecules. I haven't had any trouble with accurately measuring complex lysates using the protein-free lysate as the blank. Also I clean with .5M HCl often to recondition the pedestal, maybe every 20 uses, maybe as much as once per week. You might check with Fisher to ensure this much conditioning does not affect the metal pedestal though.

can anyone suggest me how to check labeled protein concentration by Nanodrop A280 method? I used Alexa Flour dye and want to check in vivo imaging.

Thanks!

Please have a look at this link it may help:

https://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately/.

I've read from many people about tryptophan and phenylalanine, but i don't know where is this information taken from (Tue Bjerg Bennike).

The instructions manual for Nanodrop states "The molar absorption coefficient of a peptide or protein is related to its tryptophan (W), tyrosin (Y) and cysteine (C) amino acid composition."

Manual : Protein A280P (it's a free pdf)

https://www.novoprolabs.com/tools/protein-extinction-coefficient-calculation