BRADFORD Protein Determination Protocol

Introduction and Principle

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.

Solutions/Reagents and their preparation:

• 1g Coomassie Brilliant Blue G 250 is dissolved in 50 ml 50% ethanol (v/v). After that, 100 ml of 85% phosphoric acid is added and made up with ddH2O to a total volume of 250 ml. The stock solution is stable for several months at 2–8 ◦C if prepared about 4 weeks before use.
  • Coomassie Brilliant Blue G-250 (A dye used in the binding of proteins [amazon link=”B07MFS7MQ9″ link_icon=”amazon” /] )
  • Denatured Alcohol (Ethanol) (Used in the dilutions to make buffer [amazon link=”B07BM7F8B1″ link_icon=”amazon” /])
  • Phosphoric Acid (Used in the dilutions – [amazon link=”B07CT51DRV” link_icon=”amazon” /] )
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /] )
• Dilute 1 vol. Soln. A with 4 vol. ddH2O and filter through Whatman #1 paper just before use
  • Whatman Filter Paper #1-90mm (Used for filtration purposes -[amazon link=”B0017UE2SS” link_icon=”amazon” /] )

Experiment Protocol

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.

Comments/Conclusion:

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.

Introduction and Principle: Protein Determination in SDS-PAGE Sample Solutions

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.

Solutions/Reagents:

• electrophoresis sample buffer: 62.5 mM Tris · HCl, pH 6.8, 2% SDS (w/v), 5% 2-mercaptoethanol (v/v), 10% sucrose (w/v)
  • Tris HCL (pH6.8) (An organic compound often used in buffer solutions such as TAE or TBE for electrophoresis gels -[amazon link=”B07D839R1W” link_icon=”amazon” /] )
  • Sodium Dodecyl Sulfate (It is a detergent that is used to denature proteins – [amazon link=”B07F2PP914″ link_icon=”amazon” /] )
  • 2-Mercaptoethanol [It is used to reduce disulfide linkages in solubilizing proteins for gel electrophoresis (typically used in SDS-PAGE sample buffer at 5% concentration) – [amazon link=”B07M7G78FZ” link_icon=”amazon” /] )
  • Sucrose (To reduce non-specific interactions between proteins- [amazon link=”B0771RHXXB” link_icon=”amazon” /] )
• 1M potassium phosphate buffer, pH 7.4

(Important! The use of potassium phosphate is essential!)

• • Potassium phosphate buffer (The buffer helps to maintain a constant pH – [amazon link=”B0732G7TNP” link_icon=”amazon” /] )
• dissolve 50 mg Coomassie Brilliant Blue G 250 in 50 ml ddH2O and add 50 ml 1 M perchloric acid
  • Coomassie Brilliant Blue G-250 (A dye used in the binding of proteins [amazon link=”B07MFS7MQ9″ link_icon=”amazon” /] )
  • Perchloric Acid (Used in deproteinization of proteins [amazon link=”B07MQ7L6ZK” link_icon=”amazon” /] )
• Standard: 5.0 mg/ml in 0.1% SDS (w/v)
  • Sodium Dodecyl Sulfate (It is a detergent that is used to denature proteins – [amazon link=”B07F2PP914″ link_icon=”amazon” /] )

Preparation of Reagents and Experiment Protocol:

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.

Comments/Conclusion:

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.

Introduction and Principle: Protein Determination Using Amido Black

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.

Solutions/Reagents:

• 1% Amido Black 10 B (C.I. 20470) (w/v) in 30% methanol (v/v), 70% acetic acid (v/v)
  • Amido black 10B(It is an amino acid staining azo dye used in biochemical research to stain for total protein on transferred membrane blots – [amazon link=”B074NBMJ4B” link_icon=”amazon” /] )
  • Methanol (To purify proteins – [amazon link=”B07BQYP9D1″ link_icon=”amazon” /])
  • Acetic Acid (Used as a disinfectant or a solvent in some chemical reactions [amazon link=”B078C959XB” link_icon=”amazon” /])
• methanol/glacial acetic acid 8:1 (v/v)
• 10% acetic acid (v/v) and 30% methanol in water
• 1 N NaOH
  • Sodium Hydroxide Reagent (For the dilution of reagents – [amazon link=”B00AUAB0HC” link_icon=”amazon” /] )

Preparation of Reagents and Experiment Protocol:

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.

Comments/Conclusion:

After dissolving the precipitate, measure the resulting colored solution in a photometer at 625 nm. The color of the resulting solution will be blue.

Introduction and Principle: BCA Protein Determination

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.

Procedure 1 (Standard Procedure)

Solutions/Reagents:

• 1% (w/v) BCA (2,2-biquinoline-4,4-dicarboxylic acid, bicinchoninic acid, disodium salt), 2% (w/v) Na2CO3 · H2O, 0.16% (w/v) disodium tartrate, 0.4% (w/v) NaOH, 0.95% (w/v) NaHCO3, correct pH to 11.25 if necessary with NaOH or NaHCO3. Stable at RT
  • Bicinchoninic Acid (BCA) [It is used to determine the total level of protein in a solution – [amazon link=”B07DPTY5T5″ link_icon=”amazon” /] )
  • Sodium Carbonate (Used in the dilution of reagents [amazon link=”B07MC64C66″ link_icon=”amazon” /] )
  • Sodium Tartrate (Used as a protein binding agent – [amazon link=”B0732816DL” link_icon=”amazon” /])
  • Sodium Hydroxide Reagent (For the dilution of reagents – [amazon link=”B00AUAB0HC” link_icon=”amazon” /])
  • Sodium Bicarbonate (It helps restore an optimal pH of a solution – [amazon link=”B00IOZ47JI” link_icon=”amazon” /])
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /])
• 4% CuSO4 · 5H2O. Stable at RT
  • Copper (II) Sulfate (A chemical perfect for use in any biochemistry lab – [amazon link=”B07JD2C6TW” link_icon=”amazon” /])
• Mix 100 vol. of Soln. A with 2 vol. of Soln. B. Stable at least for 1 week at RT
• Standard: 1.0 mg/ml BSA
  • Bovine Serum Albumin (BSA) (It is used as a protein concentration standard in lab experiments- [amazon link=”B07MC644FT” link_icon=”amazon” /])

Preparation of Reagents

1. Reagent A: 1 % sodium bicinchoninate (BCA), 2 % sodium carbonate, 0.16 % sodium tartrate, 0.4 % NaOH, and 0.95 % sodium bicarbonate, brought to 100 ml with distilled water. With 10 M NaOH adjust the pH to 11.25.
2. Reagent B: 0.4 % cupric sulfate (5 x hydrated) in 10 ml distilled water
3. Standard working solution (SWR): Mix 100 volumes reagent A with 2 volumes reagent B (the stock solutions should be stable and the working solution should be stable for 1 week and should be green)

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.

Procedure 2 (Micro method)

Solutions/Reagents:

1. 8% (w/v) Na2CO3 · H2O, 1.6% (w/v) NaOH, 1.6% (w/v) dis-sodium tartrate, correct pH to 11.25 with NaHCO3. Stable at RT
2. 4% (w/v) bicinchoninic acid, disodium salt (BCA). Stable at RT
3. 4% CuSO4 · 5H2O. Stable at RT
4. mix 50 vol. of Soln. A with 48 vol. of Soln. B and 2 vol. of Soln. C

Preparation of Reagents

1. Reagent A: 8 % sodium carbonate monohydrate, 1.6 % sodium tartrate, brought to 100 ml with distilled water. With 10 M NaOH adjust the pH to 11.25.
2. Reagent B: 4 % BCA in 100 ml distilled water.
3. Reagent C: 0.4 % cupric sulfate (5 x hydrated) in 10 ml of water.
4. Working solution: Mix 1 volume reagent C with 25 volumes reagent B, then add 26 volumes of reagent A to the C/B mixture.

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.

Comments/Conclusions (both procedures)

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.

References

1. Bradford, MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem;72:248-54.
2. Stoscheck, CM. (1990). Quantitation of protein. Methods Enzymol. 182:50-68.
3. Zaman, Z., Verwilghen, RL. (1979). Quantitation of proteins solubilized in sodium dodecyl sulfate-mercaptoethanol-Tris electrophoresis buffer. Anal Biochem;100(1):64-9.
4. Popov, N., Schmitt, M., Schulzeck, S., Matthies, H. (1975). [Reliable micromethod for determination of the protein content in tissue homogenates]. [Article in German: Acta Biol Med Ger; 34(9):1441-6
5. Fountoulakis, M., Juranville, JF., Manneberg, M. (1992). Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins. J Biochem Biophys Methods;24(3-4):265-74.
6. Harris, DA. CL, Bashford. (1987). Spectrophotometric assays. In, eds, Spectrophotometry and Spectrofluorimetry: a Practical Approach. IRL Press, Oxford, pp 59-61
7. K., Smith., R.I., Krohn., G.T., Hermanson et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem;150(1):76-85.