Developed by Marion M. Bradford in 1976, the Bradford assay is a quick protocol, and the same amount of proteins are used as the Lowry assay. This assay is somewhat accurate, and out-of-range samples can be retested within minutes. This assay is recommended for general use, especially for assessing protein concentrations for gel electrophoresis and determining the protein content of cell fractions.
The assay depends on the observation for an acidic solution of Coomassie Brilliant Blue G-250. The maximum absorbance shifts from 465 nm to 595 nm when binding to protein occurs. The visible color change is caused when both hydrophobic and ionic interactions stabilize the anionic form of the dye. The assay is helpful since the elimination coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.
Step 1:
Warm up the spectrophotometer before use and dilute the protein solution (standard and sample) till 10–100 µg protein at least in one assay tube.
Step 2:
Mix the solutions well after pipetting them and read the absorption at 590 nm 5 min later. The protein-dye complex is steady for a more extended period.
Note: The use of disposable plastic cuvettes is recommended. Remove adhered protein-dye complex on the walls with 96% methanol or ethanol, if glass cuvettes are used.
Determine amounts from the curve by preparing a standard curve of absorbance versus micrograms protein. From the dilution factor, amount protein, and volume/sample if any determine the concentrations of original samples.
Electrophoresis is called the separation of macromolecules in an electric field. A typical technique for isolating proteins by electrophoresis utilizes sodium dodecyl sulfate (SDS) to denature the proteins and a discontinuous polyacrylamide gel as a support medium. The method is called sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Laemmli method is the most commonly used system named after U.K Laemmli, who was the first to publish a paper in a scientific study employing SDS-PAGE.
(Important! The use of potassium phosphate is essential!)
Step 1:
In buffer, A fill 20 µl of the sample with ddH2O to 50 µl.
Step 2:
After that, 0.45 ml Soln. B is added. Vortex the solutions and centrifuge after 5–10 min with 1500−2000 × g at RT for 10 min.
Step 3:
Now, mix 2.75 ml of Soln. C with 0.25 ml of the clarified supernatant and at 620 nm read the reading in a photometer.
After taking the readings, prepare the standard curve in the range from 10 to 100 µg and 20 µl of Soln. A blank is made.
This procedure is used to measure the protein content of samples derived from primary cell cultures and cell lines. The use of trichloroacetic acid (TCA) to precipitate proteins permits removal by filtration of interfering substances in the various sample preparations; these include detergents, dyes, and reducing agents.
Step 1:
The sample is filled up to 1.0 ml with ddH2O containing up to 200 µg of protein. 2.0 ml of Soln. A is added after that.
Step 2:
Approximately for 10 minutes, the samples are put into crushed ice after mixing. Put the samples for centrifuge after that in a refrigerated centrifuge at 4 ◦C with 4000×g for 5 min.
Step 3:
Wash the pellet with Soln. B until the supernatant remains colorless and aspirate it carefully. The precipitate dries at RT after the last wash.
Step 4:
Dissolve the dry precipitate in 3.0 ml of Soln. D.
After dissolving the precipitate, measure the resulting colored solution in a photometer at 625 nm. The color of the resulting solution will be blue.
BCA reacts with complexes between copper ions and peptide bonds to produce a purple end product serving the purpose of the Folin reagent in the Lowry assay. The advantage of BCA is that it can be included in the copper solution to allow a one-step procedure and it is the reagent that is relatively stable under alkaline conditions. This method should be favored if protein concentration has to be determined in the presence of detergents. With Lowry, a molybdenum/tungsten blue product is produced.
Solutions/Reagents:
Preparation of Reagents
Experiment Protocol
Step 1:
Start the preparation of samples containing 0.2 to 50 micrograms of protein in microliters.
Step 2:
To each 20 microliters sample add 1 ml SWR and mix them. After that, incubate the sample for 30 minutes at 60 degrees C
Step 3:
After incubation, cool the samples and read the absorbances at 562 nm. For at least one hour the color will be stable.
Solutions/Reagents:
Preparation of Reagents
Experiment Protocol
Step 1:
Start the preparation of samples containing 0.2 to 50 micrograms of protein in 500 microliters.
Step 2:
To each 500 microliters sample add 500 microliters working solution and mix them. After that, incubate the mixture for 60 minutes at 60 degrees C.
Step 3:
After incubation, cool the samples and read the absorbances at 562 nm.
Prepare a standard curve of absorbance between 0 and 100 µl BSA versus micrograms (µg) protein (or vice versa), and determine amounts from the curve. From the dilution factor, amount protein, and volume/sample if any determine the concentrations of original samples.
Note: A more prolonged incubation increases the sensitivity of the assay. The heating can be stopped sooner to prevent the color from becoming too dark. The test is less sensitive and can be performed at room temperature, but there is more significant variability among proteins.
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