The estimation of proteins via the quantitative method is one of the basic requirements in biochemistry. Proteins, from various perspectives, are substantially more complex than nucleic acids. Thus, it has been hard to give laboratory protocols that can be applied to proteins. The precise quantitation of the amount of protein during the steps of protein preparation is the only valid method to check the overall value of a procedure.
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LOWRY protocol has been the most widely used method to estimate the number of proteins in biological samples. Lowry et al. (1940) was the biochemist who developed the reagent. Copper ion in alkali solution pre-treats the proteins followed by the reduction of phosphomolybdate phosphotungstic acid present in the Folin reagent. The end product of this reaction has a blue color.
Note:
This protocol is slightly modified, with respect to the original paper by Lowry, to work with smaller volumes.
Step 1:
Start the dilution of samples to an estimated 0.025-0.25 mg/ml with buffer. It is better to prepare a range of 2-3 dilutions spanning an order of magnitude if the concentration can’t be estimated. Prepare 400 microliters each dilution.
Step 2:
Prepare a reference of 400 microliters buffer. Prepare standards by adding 40-400 microliters to 13 x 100 mm tubes from 0.25 mg/ml bovine serum albumin + buffer to bring volume to 400 microliters/tube.
Step 3:
After adding 400 microliters of 2x Lowry concentrate, mix it thoroughly, and incubate at room temp. 10 min.
Step 4:
Add 0.2 N 200 microliters Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be achieved rapidly to avoid decomposition of the reagent before it reacts with protein. Incubate for 30 min. more at room temperature.
Step 5:
Use polystyrene or glass cuvettes to read the absorbances at 750 nm and after centrifugation, use an aliquot for protein determination.
Recording of absorbances should be done within 10 minutes of each other for this modified procedure. This modification is more sensitive to protein than the original and less sensitive to interfering agents. Proteins with an abnormally low or high percentage of tryptophan, tyrosine, or cysteine residues will give high or low errors, respectively.
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This modification method by Sargent (1985) is also used in the quantification of proteins. The principle of this method includes a 50-fold increase in sensitivity with respect to the Lowry standard protocol. It is possible to estimate 0.1– 1 µg protein and 4–40 µg/ml, respectively.
Step 1:
Start the dilution of samples to an estimated 0.025-0.25 mg/ml with buffer. It is better to prepare a range of 2-3 dilutions spanning an order of magnitude if the concentration can’t be estimated. Prepare 400 microliters for each dilution.
Step 2:
Prepare a reference of 400 microliters buffer and add 400 microliters of 2x Lowry concentrate, mix thoroughly, and incubate at room temp. 10 min.
Step 3:
Add 0.2 N 200 microliters Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be achieved rapidly to avoid decomposition of the reagent before it reacts with protein. Incubate for 30 min. more at room temperature.
Step 4:
Extract the samples twice with 1 ml ethyl ether each. After centrifugation remove the ether by aspiration; remove the remaining ether in the aqueous phase with a SpeedVac.
Step 5:
Now add Soln. E, i.e. Malachite green and note the observations.
Measure the absorbances at 690 nm immediately after the addition of solution E and prepare the standard curve in the range between 0 and 1 µg BSA.
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The microassay method is used for samples with low protein concentrations. The 96 well plate assay is used to perform the Lowry assay in a plate format.
Start the dilution of the sample with NaOH to a final concentration of about 0.1 moles NaOH and to an amount of protein within the measuring range.
Step 1:
Pipette 40 µl of each unknown (25 µl) and standard sample replicate into a microplate well. The same buffer that was used in the samples will be used for the diluents.
Step 2:
After adding 200 µl of freshly prepared Lowry Assay Mix, immediately mix the microplate for 30 seconds.
Step 3:
For exactly 10 minutes, incubate the microplate at room temperature.
Step 4:
After adding 20 µl of freshly prepared Lowry Working Solution to each well, immediately mix the microplate for 30 seconds.
Step 5:
Incubate 30 min at room temperature. Measure the absorbance at 620nm-660 nm on a plate reader.
If all the samples are read at the same time, the absorbance will not change significantly. The Lowry assay is not an endpoint assay, so samples will change in absorbance if too much time elapses between sample readings. During the reading of samples, i.e. less than 10 minutes, the typical time that elapses does not ordinarily result in significant changes in absorbance.
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The Lowry protein assay is a susceptible but profoundly nonspecific procedure. The standard Lowry protein assay has been adjusted so that protein can be measured in the presence of interfering chemicals. The method depends on the observation that in the presence of 125 ìg/ml of Na-deoxycholate, bovine serum albumin (5—50 ìg) is reproducibly precipitated (90–104%) by 6% trichloroacetic acid. Interference by glycerol, sucrose, EDTA, Tris—HCl, and Tricine can be eliminated. If the NaCl concentration is adjusted to 1 m, the protein samples containing carrier ampholytes can also be assayed.
Step 1:
Dilute 5 to 100 µg of protein and standard, respectively, with ddH2O to 1.0 ml.
Step 2:
After that, add 0.1 ml of Soln. A.
Step 3:
After a further 10 min at RT add 0.1 ml of Soln. B. After mixing the solution well, place the samples for centrifugation with 3000 × g at RT 15 min later.
Step 4:
Now resolve the precipitate in 1.0 ml ddH2O, and then add 1.0 ml Soln. G.
Step 5:
Now the precipitate should be resolved entirely. After further 10 minutes add 0.5 ml of Soln. H, mix well and read the absorbances at 700 nm 30–45 minutes later.
In the presence of 125 μg/ml of Na-deoxycholate at 700 nm, bovine serum albumin (5—50 μg) is reproducibly precipitated (90–104%) by 6% trichloroacetic acid.
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