8.20 Protein Assay Lab Introduction (1)
Human Biology Protein Assay Lab Introduction
Instructor Information
Dr. Laila Kudsiova
Acknowledgements: Dr. Masoud Akhtar
Institution: University of Birmingham
Learning Objectives
Become familiar with laboratory protocols for the Protein Assay before arriving for the session.
Reinforce health and safety rules in the laboratory.
Get acquainted with the equipment that will be used during the lab.
Review rules for drawing tables and graphs.
Mark Distribution
Total contribution of the lab to final grade: 30%
10% for Pre-Lab Quiz (Multiple Choice Questions online under exam conditions)
20% for Lab Report (Practical lab with write-up during a 3 hour session)
30% of the final exam mark.
Assessment Objectives
Part 1
Conduct an experiment safely following a given protocol.
Collect accurate data.
Part 2
Process data using simple mathematics/statistics.
Present data in appropriate tables/graphs.
Part 3
Draw conclusions based on collected data justified by scientific theory.
Part 4
Critically evaluate experimental procedure.
Identify potential sources of error.
Make reasonable suggestions for improvement.
Lab Rules
On Entry
Arrive on time; do not miss the demonstration.
Store coats and bags in lockers.
Silence or switch off phones.
Wear lab coats.
Be seated quietly at a workstation & await instructions.
Pay attention to the demonstration.
Bring a pen, pencil, eraser, ruler, and calculator.
No food or drink in the lab, including chewing gum.
Whilst Working
Personal safety and that of others is each individual's responsibility.
Maintain a quiet and safe working environment.
Always wear a lab coat fully buttoned up.
Wear safety glasses at all times.
Tie back long hair; avoid open-toed footwear.
Be mindful of trip hazards.
Reduce unnecessary movement; keep workspaces tidy.
On Exit
Waste disposal:
Yellow bin for biological waste.
Black bin for non-biological waste.
Clean the workbench after experiments.
Wash hands before exiting.
Lab Booklet Instructions
Treat the booklet like exam paper.
Include full name and student number.
Avoid liquid damage; all work must be performed in this booklet.
Hand the booklet to the lecturer before leaving the lab.
Individual work required, despite working in pairs in practice.
Be aware of intentional and unintentional plagiarism.
Objectives of this Lab
Determine protein concentration in two unknown samples using two different assays.
Critically evaluate the two methods of assay.
Importance of Protein Quantification
Proteins function as enzymes, hormones, and essential cellular structures.
Applications:
Diagnosing diseases through protein level measurement in blood and other fluids (e.g., low albumin indicates liver/kidney disease).
Monitoring treatment by tracking protein levels in diabetes or cancer treatment.
Research and drug development to understand protein/enzymes work, their link to disease, and potential treatments.
Manufacturing biological drugs like insulin and hormones.
Assessing nutritional values in the food industry.
Protein Quantification - Background Theory
Methods:
Colorimetric methods (binding proteins with Copper ions):
Biuret test
Lowry (Folin-Ciocalteu) assay
Bicinchoninic acid (BCA) assay
Other methods include:
High-performance liquid chromatography (HPLC)
Dye binding assay: Bradford Assay
Spectroscopy – UV Absorbance
Spectrophotometry
Both methods use spectrophotometry to measure light absorbed or transmitted by samples.
Can measure light absorbance of proteins directly or after chemical modification.
Spectrophotometer and cuvette are essential equipment.
Bradford Assay
Principle
Absorption at 595 nm is proportional to protein concentration.
Coomasie Brilliant Blue G-250 dye reacts primarily with arginine, and to a lesser extent with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues.
Transition from reddish/brown to blue at 595 nm indicates protein presence.
Procedure
Create a standard curve using known BSA concentrations for comparison against unknown samples.
Dilute standards in phosphate-buffered saline (PBS).
Record absorbance at 595 nm after adding Bradford reagent and incubating.
Create a calibration curve and interpolate unknown concentrations.
Advantages and Disadvantages
Advantages
Simple and rapid to perform.
Stable working reagent.
Broad protein concentration working range (0.2 mg/ml – 1.5 mg/ml).
Disadvantages
Not compatible with some detergents.
Need for a calibration curve.
Sensitivity varies with protein type.
UV Absorbance Assay
Principle
Aromatic amino acids in proteins absorb UV light at 280 nm.
Calculate protein concentration using the equation: [Protein] (mg/ml) = A280 x 1.55, after calibrating the spectrophotometer against a buffer blank.
Advantages and Disadvantages
Advantages
Rapid, no protein standards needed.
Sample can be recovered.
Disadvantages
Low sensitivity; affected by components that also absorb UV light.
Results influenced by pH and ionic strength.
Lab Equipment
Required Equipment
96 Well Microtiter Plate
Spectrophotometers for different absorbance measurements
Eppendorf tubes and micropipettes with various volume capacities
Protocol Steps
Experiment 1: Bradford Assay
Prepare BSA protein standards in PBS.
Add standards and samples to designated wells.
Add Bradford reagent, mix, and incubate.
Measure absorbance, calculate means, and determine concentrations using calibration curve.
Experiment 2: UV Absorbance Assay
Determine concentration of unknowns based on UV absorbance at 280 nm.
Compare concentration results with those from Bradford Assay.
Further Reading
M. Bradford (1976), Layne, E. (1957), Stoscheck, CM. (1990) studies for protein measurement methods.
Related videos on the Bradford Assay and the principle behind it.
Quick Review and Best Practices
Tables
Use pencil and ruler for clarity.
Include legends above tables and use clear labeling with units.
Graphs
Use appropriate axes and ensure legends below figures.
Ensure plots cover sufficient grid area and maintain notations.
Self-explanatory, well-structured, and referenced as necessary in text.