PART 1: Bradford Protein Assay

Materials

• Spectrophotometer set to 595 nm
• Bradford reagent purchased from Sigma-Aldrich (report the manufacturer, no recipe need required)
• Cuvettes (5 mL style)
• Eleven 13 x 100 mm test tubes and test tube rack
• 100 μL and 1000 μL pipettors and pipette tips
• BSA (bovine serum albumin) protein standard, 1.0 mg/mL
• Papain reference protein standard, 0.30 mg/mL

 Methods

1. Warm up the spectrophotometer at 595 nm for least 30 min.

2. Set up 12 clean test tubes (make sure they are not cuvette tubes) to use for the assay. You will get the best results if you mix the reaction in a large test tube and then pour a portion into the cuvette (~3 mL) for measurement.

3. Set up your protocol based on the following table. Copy the table below into your notebook. Each assay should contain 100.0 μL of water plus protein solution (either BSA, reference protein, or mushroom extract). Typically, 10–30 μl is a good range for your sample in all three colorimetric assays.

 

4. Using a micropipettor, transfer the appropriate amount of BSA standard into the tubes. For accurate pipetting, make sure the tip touches the bottom of the tube when dispensing. This also avoids risk of contaminating the Bradford reagent when you pipet it into the tube by touching the upper part of the tube's inside wall.

5. Obtain a 1 mL sample of your mushroom extract from the freezer. Choose three volumes of the unknown to assay in the range of 0–30 μL and pipet these into the last three tubes.

6. Add 3 ml of Bradford reagent to each tube. Cover the top of the tube with parafilm or a plastic cap and invert the tube a few times to mix immediately after adding the reagent to each tube. Do not wait until you have added it to all tubes.

7. Let the tubes sit for about 10 minutes before reading the absorbance. Once the color develops, it is stable for over an hour.

8. Read absorbance at 595 nm. Zero the spectrophotometer on Standard 1. To avoid the need to wash the cuvette between readings: (1) read from lowest to highest protein concentration and (2) do not decant the solution, instead completely remove the mixture with a Pasteur pipette (back into original tube).

9. In your notebook, make a rough plot to determine which BSA concentrations cause the curve to stray from linearity.

 

PART 2: Lowry Protein Assay

Materials

• Spectrophotometer set to 660 nm
• Lowry reagent (Pierce Modified Lowry Protein Assay Kit, Thermo Fisher Scientific Inc.)*
• Folin-Ciocalteu reagent (Pierce Modified Lowry Protein Assay Kit, Thermo Fisher Scientific Inc.)*
• Cuvettes (test tube-style)
• 13 x 100 mm test tubes and test tube rack
• 100 μl and 1000 μL pipettors and pipette tips
• BSA (bovine serum albumin) protein standard, 1.0 mg/ml
• Urease reference protein standard, 0.3 mg/mL

* If Lowry reagent was prepared in house, the formulation is "freshly prepared 0.20 mM copper sulfate, 51 mM sodium tartrate, 93 mM sodium carbonate, 0.50 mM sodium hydroxide" and Folin-Ciocalteu reagent was purchased from Sigma-Aldrich.

Methods

1. Set spectrophotometer at 660 nm.

2. Set-up clean tubes containing BSA standards exactly the same as in the Bradford assay. Copy the table below into your notebook.

3. Add 1.00 mL of Lowry reagent to each tube and mix. Allow to stand 10 minutes at room temperature.

4. Add 100 µL of diluted Folin's reagent (1:1). Mix immediately and let stand for 30 minutes.

5. Read absorbances at 660 nm within 30 minutes of reaction completion. Zero the spectrophotometer on Standard 1. Tubes will not be usable after one hour.

 

PART 3: BCA Protein Assay

Materials

• Spectrophotometer set to 562 nm
• BCA reagent (Pierce BCA Protein Assay Kit) (in made in house, the formulation is: 25.2 mM sodium bicinchoninate, 3.2 mM copper sulfate, 8.1 mM sodium tartrate in 300 mM pH 11.25 sodium carbonate buffer), copper sulfate added before use
• Cuvettes (test tube-style)
• 13 x 100 mm test tubes and test tube rack
• 100 μl and 1000 μL pipettors and pipette tips
• BSA (bovine serum albumin) protein standard, 1.0 mg/ml
• Urease reference protein standard, 0.3 mg/mL

 Methods

1. Set spectrophotometer at 562 nm.

2. Set-up clean tubes containing exactly the same as in the Bradford assay. Copy the table below into your notebook.

3. Add 2.00 mL of BCA reagent to each tube. Allow tubes to stand 15 minutes at 60 °C. Cool to room temperature before reading.

4. Measure absorbance at 562 nm. Zero the spectrophotometer on Standard 1. Take readings in as quick succession as possible.

 

PART 4: Spectrophotometric Protein Assay

The Nanodrop UV-Vis spectrophotometer in our lab uses a 0.1 cm pathlength so that the linear range of Beer's Law extends to an equivalent absorbance of 30 for a sample measured at a typical 1 cm pathlength. Therefore no dilution is typically necessary for most samples. Before you use the instrument, verify that the Nanodrop is set to measure 280 nm. 

1. Clean the sample pedestal with DI water and wipe dry with a Kimwipe.

2. Place 1.5 μL of phosphate buffer on the pedestal. Press Blank.

3. Wipe-off the sample pedestal with a Kimwipe, clean again with with DI water, and wipe dry.

4. Place 1.5 μL of your sample on the pedestal. Press Measure.

5. Repeat Steps 3 and 4 to measure three replicate absorbances for your mushroom extract samples and for the urease protein sample.