BIO312 – Techniques in Molecular Biology

LAB EXPERIMENT 7a: Protein Quantification viaBradford method

Background & Theory

Four spectroscopic methods are routinely used to determine the concentration of protein in a solution. These include measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; the Lowry assay, the Smith copper/bicinchoninic assay and the Bradford dye assay.

The first, UV absorbance, requires that a pure protein with known extinction coefficient be used, in a solution free of interfering (UV absorbing) substances.The Lowry and copper/bicinchoninic assays are based on reduction of Cu2+ to Cu1+ by amides. Although this makes them potentially quite accurate, they require the preparation of several reagent solutions, which must be carefully measured and mixed during the assay. This is followed by lengthy, precisely timed incubations at closely controlled, elevated temperatures, and then immediate absorbance measurements of the unstable solutions. Both assays may be affected by other substances frequently present in biochemical solutions, including detergents, lipids, buffers and reducing agents. This requires that the assays also include a series of standard solutions, each with a different, known concentration of protein, but otherwise having the same composition as the sample solutions.

The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than the assays described above. Like the other assays, however, its response is prone to influence from non protein sources, particularly detergents, and becomes progressively more nonlinear at the high end of its useful protein concentration range.The response is also protein dependent, and varies with the composition of the protein. These limitations makeprotein standard solutions necessary.

Objective

This assay is based on the use of a dye, Coomassie Brilliant Blue G-250, to which protein binds, altering the light absorbance properties of the dye. When the dye is prepared as an acidic solution (in 85% phosphoric acid), it maximally absorbs light with a wavelength of 465 nm. Addition of protein results in a shift of the dye's absorption maximum to 595 nm. As the protein concentration increases, the absorbance of light at 595 nm increases linearly. This increase in absorbance can be measured in a spectrophotometer. Although the absorbance of Coomassie blue dye at 595 nm is proportional to the amount of protein bound, it is necessary to establish a correspondence between absorbance values and known amounts of protein. To do this, you will prepare a series of protein standards – dilutions of a protein solution of known concentration. Once you have measured the A595 of each standard, you will be able to plot the A595 as a function of any unknown protein content of each standard. After measuring the A595 of unknown sample, the standard curve then can be used to determine the amount of protein corresponding to the absorbance values measured.

Protocol

REFERENCE:

  • Bradford, D, M. M. A rapid and sensitive method for the quantitaton of microgram quantities ofprotein utilizing the principle of protein-dye binding. Analytical Biochemistry, 722: p. 248-254,1976
  • Stoscheck C.M. (1990). Quantitation of protein. Methods in Enzymology 182: 50-68.

Calibration of Bradford’s curve

Reagents:

  1. BSA solution: 10mg/mL in Mili-Q water (standard BSA solution).
  2. Bradford’s reagent.

Equipment:

  1. Spectrophotometer (PerkinElmer)

Preparation of bovine serum albumin protein assay standards:

In order to measure and plot a standard curve of protein concentration versus absorbance at 595 nm, a series of dilutions of the BSA protein standard stock solution must be prepared.

The easiest way to solve for the volume of protein stock solution required for each dilution is to use the formula C1V1 = C2V2. C1 is the concentration of the protein stock solution, V1 is the volume of the stock solution required, C2 is the concentration of the diluted sample, and V2 is the volume of the diluted sample.

For example , if the concentration of the stock solution (C1) is 100 µg/ml, the concentration of the diluted sample is (C2), and the volume of the diluted sample is fixed at 200 µl. Therefore, solving for the volume of stock solution required: V1 = C2V2/C1

Protein Standards (BSA) - Protein standards should be prepared in the same buffer as the samples to be assayed. A convenient standard curve can be made using bovine serum albumin with concentrations of 10, 20, 30, 40, 50 µg/100µl for the microassay.

Bradford Reagent- Bradford reagent can be made by dissolving 100 mg Coomassie Blue G-250 in 50 ml 95% ethanol, adding 100 ml 85% (w/v) phosphoric acid to this solution and diluting the mixture to 1 liter with water.

Procedure:

  1. Prepare a 10-fold dilution of a 1 mg/ml BSA sample by adding 100 µl of 1 mg/ml BSA to 900 µl of distilled water to make 100µg/ml BSA.
  2. Generate test samples for the reference cell, blank, BSA standards and the protein sample to be tested according to Table 1 in disposable cuvettes.
  3. Note that a dilution of the protein sample may be required for the resultingabsorbance to fall within the linear range of the assay.
  4. Allow each sample to incubate at room temperature for 5 minutes.
  5. Measure the absorbance of each sample at 595 nm using a UV-visible spectrophotometer. Be sure to allow the instrument to warm up for at least 15 minutes prior to use.
  6. Plot the absorbance of each BSA standard as a function of its theoretical concentration. The plot should be linear. Determine the best fit of the data to a straight line in the form of the equation "y = mx + b" where y = absorbance at 595 nm and x = protein concentration.
  7. Use this equation to calculate the concentration of the protein sample based on the measured absorbance. If the absorbance of the test sample is outside of the absorbance range for the standards, then the assay must be repeated with a more appropriate dilution, if any. The linear range for the assay (and for most spectrophotometers is 0.2 - 0.8 O.D. units).

Preparation of BSA concentration gradient (example):

Table 1: Preparation of test samples for the Bradford protein assay.

Test Sample / Sample vol.,
µl / Water,
µl / Bradford reagent,
µl
Blank / 0 / 200 / 800
BSA Standard
(10 µg/ml) / 20 / 180 / 800
BSA Standard
(20 µg/ml) / 40 / 160 / 800
BSA Standard
(30 µg/ml) / 60 / 140 / 800
BSA Standard
(40 µg/ml) / 80 / 120 / 800
BSA Standard
(50 µg/ml) / 100 / 100 / 800
Protein Sample / 100 / 100 / 800

Read blank with distilled water and then samples (595 nm)

Coomassie dye binds to quartz cuvettes quite strongly; therefore, glass or plastic cuvettes

should be used.Warm spec for ~ 15 minutes before the assay.

LAB EXPERIMENT 7b: SDS page

Background & Theory

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate proteins with relative molecular mass no smaller than 10 KD. Very small proteins (<10 KD) are difficult to resolve due to low ability of binding to SDS, which can be solved by gradient gels or using different eletrophoresis conditions, like Tricine-SDS-page. The basic Laemmli SDS PAGE procedure is described here.

Keywords: SDS-PAGE, Protein, Electrophoresis

Materials and Reagents

  1. Pre-stain Protein MW marker (Bio-Rad Laboratories)
  2. TEMED (Life Technologies, Gibco®)
  3. Ammonium persulfate (Sigma-Aldrich)
  4. SDS (Research Organics INC)
  5. 30% Acrylamide stock (37.5: 1 acrylamide: bisacrylamide) (Bio-Rad Laboratories)
  6. Bromophenol Blue (Thermo Fisher Scientific)
  7. Tris Base (Calbiochem-Behring)
  8. Glycine (EM Science)
  9. EDTA (Amresco)
  10. Glycerol (EM Science)
  11. Isopropanol
  12. Tris-HCl (pH 6.8)
  13. β-mercaptoethanol (Sigma-Aldrich)
  14. 10x running buffer (see Recipes)
  15. 2x SDS protein sample buffer (see Recipes)

Equipment

  1. Protein mini gel cassettes (Bio-Rad)
  2. Heating block module
  3. Table-top centrifuge
  4. Power supply
  5. Gloves
  6. Filter paper

Procedure

Making SDS-PAGE gel

  1. Clean and completely dry glass plates, combs, and spacers are required.Assemble gel cassette by following manufacturer instructions.

Prepare 10% lower gel (separating gel) by adding the following solutions (wear gloves when prepare gel solution) (total volume= 5 ml)

  • 2 ml ddH2O
  • 1.67 ml 30% acrylamide/Bis
  • 1.25 ml 1.5 M Tris (pH 8.8)
  • 25 μl 20% SDS
  • 25 μl 10% ammonium persulfate (make it fresh and store at 4 °C up to a month)
  • 2.5 μl TEMED (add it right before pour the gel

Note: Change ration of ddH2O to 30% acrylamide/Bis to get different percentage of separating gel.

To avoid polymerization, after adding TEMED, mix well and quickly transfer the gel solution by using 1 ml pipette to the casting chamber between the glass plates and fill up to about 0.7 cm below the bottom of comb when the comb is in place.

Add a small layer of isopropanol to the top of the gel prior to polymerization to straighten the level of the gel.

  1. Once the gel has polymerized, start to prepare stacking gel (5%) by adding the following solutions (total volume= 3 ml)
  • 2.088 ml dH2O
  • 0.506 ml 30% acrylamide/Bis
  • 0.375 ml 1 M Tris (pH 6.8)
  • 15 μl 20% (w/v) SDS
  • 15 μl 10% ammonium persulfate
  • 1.5 μl TEMED (add it right before the gel is poured)

Remove the isopropanol layer by using filter paper. Rinse the top layer of the gel with ddH2O and dry off as much of the water as possible by using filter paper.

Add TEMED and mix the stacking gel solution well. Quickly transfer the gel solution by using a 1 ml pipette till the space is full, and then insert the appropriate comb.

Allow the top portion to solidify and then carefully remove the comb.
Note: The gels can be stored with the combs in place tightly wrapped in plastic wrap and put in a second container with wet tissue towel (keep the gels moist) at 4 °C for 1 to 2 weeks.

  1. Sample preparation
  1. Prepare same amount of protein samples according to BSA assay result, see BSA protein assay.
  2. Add the same volume of 2x protein sample buffer to each protein sample, mix and boil the samples at 95 °C heating block module for 10 min.
  3. Spin the samples at the maximal speed for 1 min (samples from some tissue/cell sources may need longer spin) in tabletop centrifuge and leave the samples at room temperature until you are ready to load onto the gel.
    Note: Can store extracted protein samples (containing sample buffer) at -20 °C and reheat at 95 °C for 5 min when used the following time.
  1. Electrophoresis
  2. Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate on the inside. Press down on the electrode assembly while clamping the frame to secure the electrode assembly and put the clamping frame into the electrophoresis tank.
  3. Pour some 1x electrophoresis running buffer into the opening of the casting frame between the gel cassettes. Add enough buffer to fill the wells of the gel. Fill the region outside of the frame with 1x running buffer.
  4. Slowly load the same amount of protein samples into each well as well as load 10 μl of protein MW marker.
  5. Connect the electrophoresis tank to the power supply.
  1. Protein detection
  2. If protein of interest is about 0.2 μg or more in the sample, typically use Coomassie blue staining (see Coomassie blue staining). Otherwise, use silver staining (sliver staining), which is more sensitive and can detect as little as 5 ng protein.

Recipes

  1. 10x running buffer
  • 30.3 g Tris-base
  • 144.0 g glycine
  • 10.0 g SDS

Completely dissolve in about 800 ml ddH2O and then more ddH2O up to 1 liter.

  1. 2x SDS protein sample buffer
  • 1.25 ml 1 M Tris-HCl (pH 6.8)
  • 4.0 ml 10% (w/v) SDS
  • 2.0 ml glycerol
  • 0.5 ml 0.5 M EDTA
  • 4 mg bromophenol blue
  • 0.2 ml b-mercaptoethanol (14.3 M)

Bring the volume to 10 ml with ddH2O.

Example of total protein SDS gel (different protocols with 2 different plant samples)

References

  1. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227(5259): 680-685.

Date: 25.12.2017

Prepared by: Jasmin Sutkovic