Another way to indirectly estimate the number of cells in a sample is to evaluate the protein content using Lowry’s method. This method has been used extensively in almost all circumstances where protein or crude extracts have been analyzed.
Quick description of the method
The method is based on a double sequence of reactions that end with the generation of color. The first is the Biuret reaction, where the peptide bonds of proteins react with copper under alkaline conditions to produce Cu+.
The Cu+ then reacts with the Folin reagent, in the Folin Ciocalteau reaction, where in substance the phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the Cu+ catalyzed oxidation.
The reactions result in a strong blue color, which depends in part on the tyrosine and tryptophan content.
The developed blue color is therefore proportional to the protein content and can be measured spectrophotometrically at 750 nm.
By creating a calibration curve using known protein concentrations as the standard (see below), it is then possible to find out the unknown protein concentration of a solution, based on the absorbance of the solution after the addition of the dye.
Some tips before starting
- The assay is performed at room temperature.
- This method is designed to quantify 1 to 20 μg of protein at a concentration that can be as low as 0.01 mg / mL. It is best used on solutions with concentrations in the range of 0.01-1 mg / ml of protein. It can be adjusted to quantify 5-100 μg of protein by increasing all volumes 5-fold.
- The test should be performed at a pH between 10 and 10.5 as it is sensitive to changes in pH.
Lowry method: Protocol
Equipment
To perform the test you’ll need pipettes and epps, a visible light spectrophotometer and polystyrene (cheap) cuvettes.
Reagents
· 0.15% (W/V) SODIUM DEOXICHOLATE (DOC)
Weigh 0.15 g of DOC and dissolve it in 100 ml of double distilled water
· 72% (W/V) TRICHLOROACETIC ACID (TCA)
Wieght 72 g of TCA and dissolve it in 100 ml of double distilled water. Store at 4C in amber bottle. Careful, it is CAUSTIC!
· COPPER TARTARATE/CARBONATE (CTC) SOLUTION
CTC stock solution
Prepare a solution 0.2% (w/v) CuSO4 . 5 H2O and 0.2% (w/v) potassium tartarate by weighting 0.1 g of aeach reagent and adding double distilled water to a final volume of 50 ml (copper solution).
Prepare a solution 20% sodium carbonate by adding 10 g of the salt to 50 ml of double distilled water.
Slowly add, while vigorously stirring, the 50 ml of the sodium carbonate solution to the first copper solution. Tis solution is stable for 2 monts at room temperature.
CTC working solution
Mix equal volumes of CTC stock solution, 0.8 M NaOH solution (0.32g of NaOH in 10 ml of water, make fresh each time), 10% (w/v) SDS solution -10 g of SDS in 100 ml – and double distelled water.
The working solution is stable for two weeks at room temperature.
· 20% (V/V) FOLIN-CIOCALTEU REAGENT
The Folin reagent (phosphomolybdic-tungstic acid) may be made by diluting (20 ml in 100 ml of double distilled water) the concentrated Folin reagent obtained from, for example Sigma, with double distilled water. This solution is stable at room temperature in an amber glass bottle
PROTEIN STANDARD
The most commonly used standards for the Lowry assay are bovine serum albumin (BSA) and bovine γ-globulin (BGG) solutions. Ovoalbumin may be used as well.
BSA 0.5 mg/ml
To prepare a 0.5 mg/ml BSA solution, weigh 5 mg BSA and dissolve it in 10 ml of double distilled H2O.
To avoid clumping, dissolve by layering the powder on the surface of the liquid while stirring. As BSA solution tends to be foamy if strongly shaken, gently rock the capped tube until the BSA has dissolved completely or stir gently with a magnetic anchor on a stirrer.
The absorbance at 280 nm of this solution in a 1-cm-path-length quarz cuvette should be 0.33 .
Store it in single-use aliquots frozen in -20 or -80 °C. Do not reuse the aliquots multiple times.
Protocol
- Make three aliquots of each of your samples by adding 5 ml, 10 ml, 25 , 50 ml, 100 ml to microcentrifuge tubes and then make up to a final volume of 500ml with double distilled water.
- Prepare calibration curve samples using 0.5 mg / mL BSA solution, create 8 BSA standards (0 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg) by diluting each up to a final volume of 500 μl with double distilled water in microcentrifuge tubes.
| µg BSA | 0 | 2 | 5 | 10 | 25 | 50 | 75 | 100 |
| µl of BSA solution (0.5 mg/ml) | 0 | 4 | 10 | 20 | 50 | 100 | 150 | 200 |
| Double distilled H2O (µl) | 500 | 496 | 490 | 480 | 450 | 400 | 350 | 300 |
3. Add 50 mL of 0.15% deoxycholic sodium to each tube and swirl by vortexing. Leave to rest for 10 min or more at room temperature..
DOC is a detergent to break cell membrane.
4. Add 50 mL of 72% TCA to each tube and vortex. Mix and leave to rest at 4 ° C 15 min. or more. At this point, the samples can be stored overnight if necessary.
TCA precipitates proteins.
5. Microcentrifuge 15 min at 3000 g at 4 ° C. Carefully remove and discard the supernatant without disturbing the TCA-protein precipitate at the bottom of the epps.
6. Dissolve pellets in 500 ul of double distilled water . Add 500 ul water to the blank tube.
7. Add 500 ml CTC working solution mix and let stand 10 min at room temperature.
An alkaline solution necessary for the formation of the complex between the Cu and the protein backbone.
8. Add 250 ml 20% (v/V) Folin-Ciocalteu reagent (color solution) and mix immediatly and very well by vortexing. Wait 30 min. for color development.
9. Turn on the spectrophotometer at 750 nm. Zero the spectrophotometer using the blank solution (0mg BSA)
10. Read the absorbance of the standard and sample(s) at 750 nm.
11. Record the A750 values and plot your standard curve (log A750 vs. log mg BSA) and determine the amount of protein (mg) in each of your samples by extrapolation from the standard curve.
Use only data points that fall within the range of the standard curve.
Calculate the protein concentration (mg / mL) of each of your samples.
Conclusive remarks
Many substances interfere with protein determination by this method (Peterson 1979). Some of the commercially available buffers produce the blue color (with the same absorption spectrum of the dye produced by protein) in the absence of protein (HEPPS, HEPES, and Bicine, and to a much lesser degree BES, PIPES, ADA, ACES, MOPS, glycineamide, TAPS, and CAPS). Some of the buffers, in addition to giving color, prevent the formation of the normal amount of color by proteins (ADA, Tris, Tricine, and TAPS).
If detergents, denaturants, organic buffers and thiols are contained in the protein solution under examination, it is important to determine their effect on absorbance, creating a calibration curve in the presence of these compounds.
However, it must be said that, unlike the Bradford, the precipitation of proteins via DOC / TCA will eliminate many of these interfering substances.
The A750 decreases at a rate of 1%, 2% per hour at room temperature.
If the test protein sample contains detergents such as 1% tryto, Tween, NP-40 or 0.75% cetyltrymethyl ammonium bromide, adding 4% SDS prevents precipitation of the Folin reagent