First you have to select the best method for measuring protein, according to sample type and presence of substances as potential interferences.

The Bradford method, Lowry method, Absorbance at 280 nm method, have their advantages and disadvantages.

Bradford method is fast, sensitive, accurate, has low interferences.

The absorbance at 280 method is fast, requires no chemicals, is non-destructive, but is low sensitive and inaccurate.

The method of Lowry has many steps, the color is dependent on the incubation time, it is less sensitive than Bradford and has a lot of interference with common substances.

In any case you must construct a standard curve (using bovine albumin).

For that you have to prepare a stock solution of albumin, and from this prepare different solutions of known concentration.

These albumin solutions are used as samples, applying the selected method, to construct the standard curve.

Graph Absorbance vs concentration, and obtains the equation of the line (y = mx + b), with r2, as close to 1 as possible.

Another option is to subtract the value of "zero" protein, then the straight line passes through zero and the equation simplifies: y = mx.

Where y is the absorbance, m is the slope and x is the concentration.

Thus, x = y / m.

The absorbance of the sample is divided by the value of the slope and the concentration is obtained.

If the sample was diluted, the value is adjusted by multiplying by the dilution factor.

Example:

Absorbance = 0. 36

Sample was diluted 10 times

The absorbance of the "zero" is 0.053

Adjusted absorbance: 0. 360-0.053 = 0.307

The standard curve equation: y = 0.012x

x = y / m,  therefore x = 0.307 / 0.012

x = 25.58 ug / ml

But sample diluted 10 times:

x = 25.58 x 10 = 255.8 ug prot / ml

Compare to a standard curve?

I.e. make a solution of known protein concentration (usually using some fairly cheap, high purity bulk-protein like BSA) and prepare a serial dilution series covering the expected range of your test samples, and then use the lowry method on those to establish a relationship between OD and [protein].

This page

http://www.chem.ucla.edu/dept/Faculty/merchant/pdf/Lowry_Assay.pdf

summarises it pretty well.

Karim, which protein are you working with? 

You could always use a nanodrop to calculate protein concentration by taking the absorbance reading at 280, you do not need to make a standard curve for this and it is probably the fastest (however not most accurate) way to calculate protein concentration in a purified sample.  http://www.jove.com/video/1610/microvolume-protein-concentration-determination-using-nanodrop-2000c

Thanks a lot Fabian, I use Nanodrop and absorbance at 750.

absorbance at 750 is still for Lowry. standard curve (generally BSA is readily available and useful) is really best way to determine protein concentration accurately. however, an additional note, make sure to measure diluent alone too. Lowry can react with some standard protein prep solutions, such as RIPA

First you have to select the best method for measuring protein, according to sample type and presence of substances as potential interferences.

The Bradford method, Lowry method, Absorbance at 280 nm method, have their advantages and disadvantages.

Bradford method is fast, sensitive, accurate, has low interferences.

The absorbance at 280 method is fast, requires no chemicals, is non-destructive, but is low sensitive and inaccurate.

The method of Lowry has many steps, the color is dependent on the incubation time, it is less sensitive than Bradford and has a lot of interference with common substances.

In any case you must construct a standard curve (using bovine albumin).

For that you have to prepare a stock solution of albumin, and from this prepare different solutions of known concentration.

These albumin solutions are used as samples, applying the selected method, to construct the standard curve.

Graph Absorbance vs concentration, and obtains the equation of the line (y = mx + b), with r2, as close to 1 as possible.

Another option is to subtract the value of "zero" protein, then the straight line passes through zero and the equation simplifies: y = mx.

Where y is the absorbance, m is the slope and x is the concentration.

Thus, x = y / m.

The absorbance of the sample is divided by the value of the slope and the concentration is obtained.

If the sample was diluted, the value is adjusted by multiplying by the dilution factor.

Example:

Absorbance = 0. 36

Sample was diluted 10 times

The absorbance of the "zero" is 0.053

Adjusted absorbance: 0. 360-0.053 = 0.307

The standard curve equation: y = 0.012x

x = y / m,  therefore x = 0.307 / 0.012

x = 25.58 ug / ml

But sample diluted 10 times:

x = 25.58 x 10 = 255.8 ug prot / ml

It is not possible to estimate the protein content afterwards without having a series of known protein standard. I used Nanodrop (3 machines on campus) serveral times myself and got very strange results when standards were not available. I will not even use a standard curve obtained after this OD is obtained since the reaction time and condition will be different. So, go back to drawing board and measure it together with the protein standards.

I agree with others that Nanodrop method is inaccurate and it depends on the aromatic amino acid content. 

I have used the Lowry method for years on a number of different type of samples without any problems.  Just be sure to run a reagent blank.  What I have not seen in the above answers is to be sure to check the concentration of your BSA standard by measuring the A280 and then calculating using the extinction coefficient for BSA. (1mg/ml BSA, 1cm light path, A280=0.660).

.

I have heard a strange calculation.

• Read the OD for sample
• Subtract BLANK OD, now you have net OD
• Then, multiply this net OD with dilution factor
• Then, calculate the protein concentration using standard curve

What do you say? I think it is wrong !... If I have already developed the final colored substance or complex that I am about the read OD, it will not be accurate if I dilute this final product with a diluent and read OD and calculating the protein concentration.

Dilution is related to protein sample. Is not releted to colured solution, no.

If your protein concentration is to high the color developed is also high, then absorbance is out of your standard curve. You must dilute your sample and repeat the assay.

Yours

Hector Nolasco

Now , we have settled that the dilution if for protein sample :-)

Best

You meant mg/ml? If so, 1mg= 1000 ug. Volume does not change. 100ug/ml protein is equal to 0.1 mg/ml protein

You need to calculate  the volume of the solution in which you have processed known amount of leaf. Say, you prepared your 1 g leaf extract in 10 ml of buffer. The protein concentration was  100 ug/ml (=0.1 mg protein/ml). But your protein stock solution is 10 ml, hence  10 X 0.1 mg = 1 mg total protein in 10 ml solution; therefore your protein concentration in 1 g leaf would be 1 mg protein / g leaf.

Hope it helps :-)

 Baki

I think this link is very useful, 

http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/

I guess you are intending to use Bradford reagent for protein determination as you are writing about measuring Abs at 595nm... Working with 1mL of Biorad Bradford Reagent, BSA samples for calibration shouldn't contain more than 20µg in 20µL maximum. So 1mg/mL BSA solution is OK. For drawing calibration curve, prepare different cuvettes containing 1mL of Bradford Reagent in which you will add different volumes of the 1mg/mL BSA solution (0, 5, 10, 15, 20 µL for instance to get 0, 5 10 15 20 µg) and mix. After 5' incubation at room temperature, read at 595nm with setting zero with the first tube. Abs=f(µg BSA) should be linear. This calibration curve will allow you to determine the content in protein of any sample submitted to Bradford assay. And knowing the volume used for the assay you will be able to calculate the protein solution concentration.

Mohamed G shehata, Sir.. We will make stock Solution of 1 mg/ml... Then we will take 5, 10, 15, 20 microlitter seperately from the stock and mix into different cuvett containg 1 ml bradford reagent, my doubt is that we will take 1 ml of bradford reagent in each vial or we will take 995 microlit, 990 microlit, 985, n 980 microlit of bradford reagent and then???

Sir if we want to perform protein estimation in 96 well plate. Then we will transfer the solution from cuvet to 96 well plate or consider any other factor??

Correct, add 1 ml of Bradford reagent in all tubes and read the absorbance. For 96 well plate, there is aseparate protocol for 96 well plate format. Basically, you proportinally reduce to 250 ul (the volume of microplate well). This method alsp saves reagent; however, you can perform the color development in 1 ml tube and just before reading, mix the tubes well, and get , say, 200 ul from the tube, place in 96 well plate and read.

Hope it helps

I am trying to measure the concentration of my protein encapsulated in MOFs by using Lowry's method. However the method is showing far higher protein concentration (100 mug/ml) in the supernatant than I actually used (10 mug/ml). Can anybody please elaborate upon the possible reason?

Use Bradford method, and construct a standard curve, using bovine albumin.

H.

Dear Hector,

Thanks much for your suggestion.

Best regards,

Deepak

Some one can explain what is the K-value and how is it determined, in Lowery method used for leaf protein content?

Here are some comparison among the various protein estimation method. Hope that Deepak will find the answer .

Best,

Baki

hi, I am using 1ml culture of yeast for detecting cellular proteins by Lowry method. So i have to calculate total protein contents. can anyone suggest me formula to calculate protein contents? Moreover m using 200ul of buffer to dissolve proteins.

I am estimating protein content in fish tissues by kjeldahl method..

.protein =%nitrogen ×6.25

But my answers are not feasible as per literature..can anybody help me in calculation part

Azra Bashir Please see the following file and hopefully it can help you in your research.

http://aquaticcommons.org/23107/1/IFRO-v16n2p668-en.pdf

https://promega.media › application-notes

Here in protein calculation dilution means complete stock buffer solution in which the sample was processed?