Protein Quantification Protocols

Introduction: Quantitative Method

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.

LOWRY Protein Quantification

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:

• An aliquot of the protein-free buffer in the same volume as the protein-containing sample has to be taken as blank as a control.
• A couple of standards with different amounts of protein between 0 and 100 µg should be measured in each analysis since the reaction conditions may differ from experiment to experiment and the standard curve is not linear.

Solutions/Reagents:

• 20 g Na2CO3 (anhydrous) in 1000 ml 0.1 N NaOH
  • Anhydrous Sodium Carbonate (Used in the dilution of reagents – [amazon link=”B07MC64C66″ link_icon=”amazon” /])
  • Sodium Hydroxide Reagent (For the dilution of reagents – [amazon link=”B00AUAB0HC” link_icon=”amazon” /] )
• 1g CuSO4 · 5H2O in 100 ml ddH2O
  • Copper (II) Sulfate (A chemical perfect for use in any biochemistry lab – [amazon link=”B07JD2C6TW” link_icon=”amazon” /] )
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /] )
• 2g potassium-sodium tartrate in 100 ml ddH2O
  • Potassium Sodium Tartrate( It is a common precipitant in protein crystallography and maintains cupric ions in solution at an alkaline pH – [amazon link=”B07MJDDXWW” link_icon=”amazon” /])
• mix 1 vol. B and 1 vol. C, and then add 50 vol. A
• Folin–Ciocalteu’s phenol reagent (stock), 1 + 1 diluted with ddH2O
  • Folin–Ciocalteu’s phenol reagent(Reagent used in LOWRY method – [amazon link=”B07MJDGBT9″ link_icon=”amazon” /] )
• Standard 5.0 mg/ml ovalbumin or BSA, 0.1% SDS (w/v) in ddH2O
  • 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. Dissolve 0.4 gm cupric sulfate (5x hydrated) in 20 ml water, 20 gm sodium carbonate in 260 ml water, and 0.2 gm sodium potassium tartrate in 20 ml water. Prepare the copper reagent by mixing all three solutions.
2. Prepare the solution of sodium dodecyl sulfate (SDS): 100 ml of a 1% solution (1 gm/100 ml)
3. Prepare a solution of NaOH: 1 M (4 gm/100 ml)
4. Mix 1 part NaOH with 3 parts copper reagent and 1 part SDS for the 2x Lowry concentrate. Warm the solution to 37 degrees if a white precipitate forms, and discard if there is a black precipitate.
5. Mix 10 ml 2 N Folin reagent with 90 ml water to prepare 0.2 N Folin reagent.

Experiment Protocol

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.

Comments/Conclusion:

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.

Introduction and Principle: Modification by SARGENT

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.

Solutions/Reagents:

• 20 mM CuSO4, 40 mM citric acid, 0.1 mM EDTA
  • Copper (II) Sulfate (A chemical perfect for use in any biochemistry lab – [amazon link=”B07JD2C6TW” link_icon=”amazon” /] )
  • Citric Acid (It is an intermediate in the citric acid cycle, which occurs in the metabolism of all aerobic organisms – [amazon link=”B00EYFKKZC” link_icon=”amazon” /] )
  • Ethylenediaminetetraacetic acid (EDTA) (Water-soluble solid used to prepare solutions – [amazon link=”B07MJDDPQK” link_icon=”amazon” /])
• 4 M Na2CO3, 0.32 M NaOH
  • Anhydrous Sodium Carbonate (To make the dilute solutions – [amazon link=”B07MC64C66″ link_icon=”amazon” /] )
  • Sodium Hydroxide Reagent (For the dilution of reagents – [amazon link=”B00AUAB0HC” link_icon=”amazon” /])
• mix 1 vol. freshly prepared A with 25 vol. freshly prepared B
• Folin–Ciacalteu’s phenol reagent (stock), 1 + 1 diluted with ddH2O
  • Folin & Ciocalteu’s Phenol Reagent (Reagent used in LOWRY method – [amazon link=”B07MJDGBT9″ link_icon=”amazon” /])
• 60 µg/ml malachite green in 0.1 M sodium maleate buffer, pH 6.0, 1 mM EDTA
  • Malachite Green (Chemical for general purpose lab and used as a staining agent – [amazon link=”B07JMMQJ3J” link_icon=”amazon” /] )
  • Ethylenediaminetetraacetic acid (EDTA) (Water-soluble solid used to prepare solutions – [amazon link=”B07MJDDPQK” link_icon=”amazon” /] )

Preparation of Reagents

1. Prepare a 20 mM solution of copper sulfate and a 40mM solution of citric acid in 0.1 mM EDTA. It is recommended to make samples, blank, and standards at least in duplicates.
2. Prepare a 0.40 M solution of sodium carbonate
3. Prepare a 0.32 M solution of NaOH
4. Prepare 0.2 N Folin reagent by mixing 10 ml 2 N Folin reagent with 90 ml of water
5. Prepare 60 µg/ml malachite green in 0.1 M sodium maleate buffer

Experiment Protocol

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.

Comments/Conclusion:

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.

Introduction and Principle: Micromethod on Microtest Plates

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.

Solutions/Reagents:

• 20 g Na2CO3 (anhydrous) in 1000 ml 0.1 N NaOH
  • Anhydrous Sodium Carbonate (To make the dilute solutions -[amazon link=”B07MC64C66″ link_icon=”amazon” /] )
  • Sodium Hydroxide Reagent (For the dilution of reagents – [amazon link=”B00AUAB0HC” link_icon=”amazon” /])
• 0g CuSO4 · 5H2O in 100 ml ddH2O
  • Copper (II) Sulfate (A chemical perfect for use in any biochemistry lab – [amazon link=”B07JD2C6TW” link_icon=”amazon” /])
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /] )
• 0g potassium-sodium tartrate (Seignette salt) in 100 ml ddH2O
  • Potassium Sodium Tartrate (It is a common precipitant in protein crystallography and maintains cupric ions in solution at an alkaline pH – [amazon link=”B07MJDDXWW” link_icon=”amazon” /] )
• mix 1 vol. B and 1 vol. C, and then add 50 vol. A
• Folin–Ciocalteu’s phenol reagent (stock), 1 + 1 diluted with ddH2O
  • Folin & Ciocalteu’s Phenol Reagent (Reagent used in LOWRY method – [amazon link=”B07MJDGBT9″ link_icon=”amazon” /] )
• Standard 2.0 mg/ml BSA in 0.1 N NaOH (stable at 2–8 ◦C for several months)
  • Bovine Serum Albumin (BSA) (It is used as a protein concentration standard in lab experiments – [amazon link=”B07MC644FT” link_icon=”amazon” /] )

Preparation of Reagents

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.

Experiment Protocol

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.

Comments/Conclusion:

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.

Introduction and Principle: Protein Determination in the Presence of Interfering Substances

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.

Solutions/Reagents:

• 15% sodium deoxycholate (w/v) in ddH2O Solutions/Reagents
  • Sodium Deoxycholate (It is an ionic detergent for solubilizing and isolating membrane-bound proteins in an active state – [amazon link=”B0771S29QQ” link_icon=”amazon” /])
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /] )
• 72% trichloroacetic acid (w/v) in ddH2O
  • Trichloroacetic Acid (It is widely used in biochemistry for the precipitation of macromolecules such as proteins – [amazon link=”B07F5MKR4Q” link_icon=”amazon” /])
• 1% CuSO4 (w/v) in ddH2O
  • Copper (II) Sulfate (A chemical perfect for use in any biochemistry lab – [amazon link=”B07JD2C6TW” link_icon=”amazon” /] )
• 2% sodium-potassium tartrate (w/v) in ddH2O
  • Potassium Sodium Tartrate (It is a common precipitant in protein crystallography and maintains cupric ions in solution at an alkaline pH – [amazon link=”B07MJDDXWW” link_icon=”amazon” /] )
• 3.4% sodium carbonate (anhydrous) (w/v) in 0.2 N NaOH
  • Anhydrous Sodium Carbonate (To make the dilute solutions – [amazon link=”B07MC64C66″ link_icon=”amazon” /])
  • Sodium Hydroxide Reagent (For the dilution of reagents – [amazon link=”B00AUAB0HC” link_icon=”amazon” /] )
• 10% SDS (w/v) in ddH2O
  • 10% SDS (Sodium Dodecyl Sulfate) (It is a detergent that is used to denature proteins – [amazon link=”B017MX7EXI” link_icon=”amazon” /] )
• Mix just before using Soln. C, D, E, and F in a ratio of 1:1:28:10
• Folin–Ciocalteu’s phenol reagent (stock), diluted 1 + 3 with ddH2O
  • Folin–Ciocalteu’s phenol reagent (Reagent used in LOWRY method – [amazon link=”B07MJDGBT9″ link_icon=”amazon” /] )

Preparation of Reagents and Experiment Protocol:

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.

Comments/Conclusion:

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.

References

1. Lowry, OH., NJ, Rosbrough., AL, Farr., and RJ, Randall. J. (1951). Protein measurement with the Folin phenol reagent. Biol. Chem;193(1):265-75.
2. Sargent, MG. (1987). Fiftyfold amplification of the Lowry protein assay. Anal Biochem; 163(2):476-81.
3. Oosta, GM., Mathewson, NS., Catravas, GN. (1978). Optimization of Folin–Ciocalteu Reagent concentration in an automated Lowry protein assay. Anal Biochem; 89(1):31-4.
4. Stoscheck, CM. (1990). Quantitation of protein. Methods Enzymol. 182:50-68.
5. Hartree, EF. (1972). Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem; 48(2):422-7.
6. 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.