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2: Activity 1-2 - Bradford Assay – Measuring Protein Concentration

Last updated

Apr 19, 2025
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- 1: Activity 1-1 - Protein Extraction Using Bacterial Cell Lysis
- 3: Activity 1-3 - Beer’s Law Spectrophotometric Analysis

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Learning Objectives

Explain the principle of the Bradford protein assay and how it detects protein concentrations.
Understand why measuring protein concentration is essential before assessing enzyme activity.
Describe the structure and function of cytochrome P450 enzymes, particularly P450_BM3.
Identify the significance of using a standard curve in quantitative spectrophotometry.
Prepare reagents and samples accurately using serial dilution techniques.

Definition: Term

Bradford Assay: A colorimetric assay that uses Coomassie Brilliant Blue G-250 to detect and quantify protein concentrations.
Coomassie Brilliant Blue: A dye that binds to proteins, especially basic and aromatic residues, causing a shift in absorbance from 465 nm to 562 nm.
Standard Curve: A graph of known concentrations used to determine the concentration of unknown samples.
Cytochrome P450 (P450s): A family of heme-containing enzymes that catalyze oxidation of substrates including drugs and toxins.
P450_BM3: A self-sufficient bacterial cytochrome P450 enzyme from Bacillus megaterium, containing fused reductase and heme domains.
Absorbance (A): A measure of the amount of light absorbed by a solution at a specific wavelength.
Cell-Free Extract (CFE): A lysate containing soluble cellular components, including proteins, from which intact cells have been removed.
Dilution Factor: The ratio by which a solution is diluted; in this lab, samples are diluted 10-fold.

Pre-Lecture Questions

What are the advantages of using the Bradford assay for protein quantification in a complex sample like a cell-free extract?
Why do we use BSA as a standard in protein assays?
What role does the heme group play in cytochrome P450BM3 activity?
How does the structure of P450BM3 make it unique compared to other P450 enzymes?
Why is it important to determine the protein concentration before performing enzyme activity assays?

Note

Prepare a dilution series of BSA to generate a standard curve.
Perform the Bradford assay on CFE-A and CFE-B.
Use a spectrophotometer to measure absorbance at 562 nm.
Plot a standard curve and determine unknown protein concentrations.

Week 1b: Bradford Assay – Measuring Protein Concentration

Cytochrome P450 enzymes are a large and diverse family of heme-containing monooxygenases that play critical roles in the metabolism of drugs, toxins, and endogenous compounds across all domains of life. In humans, these enzymes are involved in detoxification and drug metabolism in the liver, while in microorganisms, they often help degrade environmental chemicals or participate in specialized metabolic pathways. Because of their catalytic versatility, cytochrome P450s are widely studied in biotechnology for applications such as drug development, bioremediation, and synthetic biology. In this lab, we focus on P450_^BM3 from Bacillus megaterium, a well-characterized bacterial P450 that is especially useful for educational and research purposes. Unlike most P450s that require separate reductase proteins, P450BM3 is a naturally fused, self-sufficient enzyme consisting of a 119 kDa polypeptide with FAD, FMN, and heme cofactors all in one protein. This fusion allows for efficient electron transfer from NADPH to FAD, then to FMN, and finally to the heme center, where oxygen activation and substrate oxidation occur. The heme group binds oxygen and substrate, producing a characteristic absorption peak at 418 nm, a spectral signature used to detect the presence of active P450_BM3.

For this week’s experiments, two cell-free extracts have been prepared from E. coli: one expressing an empty vector plasmid (pET3) and the other expressing the P450_BM3 gene (pT7_BM3). Only the extract containing P450_BM3 will show characteristic P450 activity. To investigate this, students will perform three spectrophotometric assays: (1) the Bradford assay to estimate total protein concentration using Coomassie Brilliant Blue G-250 dye, which shifts absorbance from 465 nm to 562 nm when bound to proteins—mainly through interactions with basic and aromatic residues; (2) an absorption scan to detect the 418 nm peak associated with the heme group of cytochrome P450_BM3; and (3) an enzyme activity assay to monitor NADPH oxidation by measuring the decrease in absorbance at 340 nm.

The Bradford assay will provide an estimate of protein concentration in the extracts, although it does not distinguish between P450BM3 and other cellular proteins. The combined results will help students visualize and quantify the presence and activity of a bacterial cytochrome P450 enzyme, laying the foundation for understanding how these enzymes function and why they are important in both natural and applied contexts.

In this lab, you will use the Bradford assay to estimate protein concentrations in two cell-free extracts (CFE-A and CFE-B) using a standard curve generated from known concentrations of bovine serum albumin (BSA). This is an essential first step in protein studies and helps us normalize enzyme activity in later experiments.

Materials

Pierce BCA Protein Assay Kit (Thermo Scientific, #23227)
- (Contains BSA standard [2 µg/µL], Reagent A, and Reagent B)
Plastic cuvettes (at least 10)
Distilled water
Cell-free extracts A and B (from previous lab)
15-mL or 50-mL conical tubes
37°C incubator
Spectrophotometer
Pipettes and tips
Parafilm (to seal cuvettes)
Scissors, Sharpies, acetone, cotton swabs

Protocol

Remove the "Bradford" tubes containing CFE-A and CFE-B from the freezer and allow them to thaw on ice.
In a 15-mL conical tube, mix 5 mL of Reagent A with 100 µL of Reagent B (this is a 50:1 ratio).
Label this tube “Bradford Reagent” and keep it at room temperature.
Obtain 10 cuvettes
- 2 cuvettes: one each for "CFE-A" and "CFE-B"
- 6 cuvettes: for your "BSA standard" (#1 to #6)
- 1 cuvette: for the "blank" (contains no BSA)
- 1 extra in case of mistakes

Prepare the BSA standard curve (see Table 1)

Use the serial dilution method to generate known concentrations:
- Label the "BSA standard" cuvettes #1 through #6.
- Add 100 µL of BSA standard (2 µg/µL) to cuvette #1.
- Add 50 µL of water to cuvettes #2 to #6.
Mix as follows:
- Transfer 50 µL from cuvette #1 to #2, mix well.
- Then take 50 µL from #2 to #3, mix.
- Continue: #3 to #4, then #4 to #5.
- Discard 50 µL from cuvette #5 after mixing. (DO NOT add anything yet to #6).
- Cuvette #6 is your zero point (blank); it only receives water.

You now have a dilution series with decreasing concentrations of BSA from cuvette #1 to #5.

Prepare the samples (see Table 2)

Dilute each of your cell-free extracts, labeled CFE-A and CFE-B
- 5 µL of your sample + 45 µL of water (this is a 10-fold dilution)

Bradford Assay

To each cuvette (standards, samples, and blank), add 450 µL of Bradford reagent
Immediately, seal with Parafilm and invert gently a few times to mix (do not vortex!)
Place all cuvettes in a 37°C incubator.
Incubate for at least 10 minutes (you can go up to 1 hour for deeper color development).
After incubation, use a spectrophotometer set to 562 nm and measure the absorbance of each standard, sample, and blank.

Create your standard curve

On Excel or Sheets, plot:
- X-axis: BSA concentration (µg/µL)
- Y-axis: Absorbance at 562 nm
Add a trendline and display the equation and R² value.
Use the equation to calculate the protein concentration in CFE-A and CFE-B, remembering to account for the 10-fold dilution.

Tables for Reference

Table 1 – BSA Standard Curve Setup

Cuvette	BSA (µL)	H₂O (µL)	Bradford Reagent (µL)	Final BSA (µg/µL)
#1	50	0	450	2.00
#2	25	25	450	1.00
#3	12.5	37.5	450	0.50
#4	6.25	42.75	450	0.25
#5	3.125	46.875	450	0.125
#6 (Blank)	0	50	450	0.00

Table 2 – Diluted Sample Setup

Cuvette	Dilution	Sample (µL)	Water (µL)	Bradford Reagent (µL)	Absorbance (562 nm)
CFE-A	10-fold	5	45	450
CFE-B	10-fold	5	45	450

Learning Objectives

Accurately interpret a standard curve to calculate protein concentrations.
Evaluate the reliability of spectrophotometric data using R² values from trendlines.
Reflect on the limitations of the Bradford assay (e.g., protein-to-protein variability).
Discuss why accurate protein quantification is foundational for normalizing enzymatic activity.
Connect the Bradford assay to future assays detecting cytochrome P450 function (absorption at 418 nm and NADPH consumption at 340 nm).

Key Reflections

Prompt	Student Response Example
What did the standard curve tell you about your samples?	“It helped us estimate the protein concentration of CFE-A and CFE-B based on their absorbance.”
How reliable was your data (based on R² value)?	“Our R² value was 0.99, suggesting a strong linear relationship between BSA concentration and absorbance.”
Did both CFEs show similar protein concentrations? Why might they differ?	“They were different, possibly because only one sample expresses the P450_BM3 protein.”
What limitations might affect the Bradford assay’s accuracy?	“It measures total protein, so it doesn’t tell us how much of that protein is P450_BM3.”
How will knowing protein concentration help in later P450 assays?	“We need to normalize enzyme activity to protein amount to compare true enzyme efficiency.”

Post-Lecture Questions

What wavelength is used to measure the Bradford assay and why?
How would you troubleshoot if your blank cuvette had an unexpectedly high absorbance?
If two extracts have different protein concentrations, how might that affect their enzyme activity measurements?
What does an R² value of 0.85 tell you about your standard curve?
In the context of drug metabolism, why is it important to measure the expression and activity of enzymes like cytochrome P450?

Learning Objectives

Definition: Term

Pre-Lecture Questions

Note

Week 1b: Bradford Assay – Measuring Protein Concentration

Materials

Protocol

Prepare the BSA standard curve (see Table 1)

Prepare the samples (see Table 2)

Bradford Assay

Create your standard curve

Tables for Reference

Table 1 – BSA Standard Curve Setup

Table 2 – Diluted Sample Setup

Learning Objectives

Key Reflections

Post-Lecture Questions

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