The Arizona STEM Acceleration Project
Protein Assay Using Serial Dilutions lab
Protein Assay Using Serial Dilutions Lab
A 11th and 12th grade STEM lesson
Miranda Thornton
July 2024
Notes for teachers
List of Materials
Standards
HS+B.L3U1.11 Construct an explanation for how the structure of DNA and RNA determine the structure of proteins that perform essential life functions.
HS.P4U1.10 Construct an explanation about the relationships among the frequency, wavelength, and speed of waves traveling in various media, and their applications to modern technology.
Standards
STEM STandards:
MathQR.SPR.3: Represent numerical summaries and visual displays of real-world data to make informed decisions. Reason, communicate, and describe strengths, limitations, and fallacies of various displays. Encompasses P.S-IC.B.6
A1.F-IF.C.7 Graph functions expressed symbolically and show key features of the graph, by hand in simple cases and using technology for more complicated cases. Focus on linear, quadratic, exponential and piecewise-defined functions (limited to absolute value and step
Bioscience standards:
1.1 Identify and wear appropriate lab attire and personal protective equipment (e.g., safety glasses or goggles, lab coat, gloves, and closed-toe shoes)
9.4 Perform protein assays and compare to protein standards (i.e., Bradford and Lowry methods, etc.)
Objective(s):
Today: After this activity students should be able to:
Definitions
Bradford Assay: The Bradford method is a quantitative protein assay method, based on the binding of a dye, Coomassie Brilliant Blue, to a protein sample, and comparing this binding to a standard curve generated by the reaction of known amounts of a standard protein, usually BSA.
Serial Dilutions: A serial dilution is the step-wise dilution of a substance in solution, either by using a constant dilution factor, or by using a variable factor between dilutions. If the dilution factor at each step is constant, this results in a geometric progression of the concentration in a logarithmic fashion.
Agenda (lesson time)
Today:
Give students that driving question(What are the ways you can test the protein in the sample that you have?)
This will take about 4 days maybe 5
Make sure to go over the procedures and where they can get their materials for the lab.
Definitely have them pick roles within their group.
Driving Question
What are the ways you can test the protein in the sample that you have?)
How to determine the Protein Concentration with the Bradford Assay (youtube.com)
Protein Quantification (Bradford Assay) (youtube.com)
Hands-on Activity Instructions
Think about the following question:
1. what are the ways we can test the concentration of a protein?
Hands-on Activity
Instructions
Making the Standard Curve:
As outlined below, prepare a 2-fold serial dilution of the Bovine Serum Albumin (BSA)
in water from 1.0 mg/mL down to 0.062 mg/mL (62 µg/mL) – BSA stock is 2 mg/mL
1. Label six microcentrifuge tubes with corresponding protein concentrations (1.0
mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.062 mg/mL and 0 mg/mL)
2. Add 100 µL of dH2O to each tube
3. Add 100 µL of 2 mg/mL BSA stock to the tube labeled “1.0 mg/mL”
4. Mix gently by pipetting up and down 5 times
5. Remove 100 µL from the 1mg/mL tube, and add it to the 0.5 mg/mL labeled tube
6. Mix by pipetting, then continue serial dilutions down to 0.062 mg/mL
7. Do not add anything to the final tube containing water (this will be “0” mg/mL)
Hands-on Activity Instructions
Preparing the Protein Sample
Prepare a 4-fold serial dilution for your protein sample as follows:
Label 5 microcentrifuge tubes as: 1:4, 1:16, 1:64, 1:256, 1:1024.
1. Add 750 µL dH2O to each of the five tubes.
2. Add 250 µL of your protein sample into its corresponding, labeled 1:4 tubes. Mix
gently by pipetting up and down 5 times
3. Remove 250 µL from the 1:4 dilution and add to the corresponding 1:16 tubes.
Mix gently by pipetting up and down 5 times
4. Continue serial dilutions down to the 1:1024 tube. Mix gently by pipetting up and
down 5 times
You should now have five tubes of your protein sample diluted 1:4, 1:16, 1:64,
1:256, and 1:1024.
Hands on activity
Instructions
Performing the Assay
Using a 96-well plate, add the standards and samples to the corresponding wells as
follows: (See diagram on page 3).
1. Add 25 µL of the 1 mg/mL Standard to wells A-1 through A-3
2. Add 25 µL of the 0.5 mg/mL Standard to wells B-1 through B-3
3. Add 25 µL of the 0.25 mg/mL Standard to wells C-1 through C-3
4. Add 25 µL of the 0.125 mg/mL Standard to wells D-1 through D-3
5. Add 25 µL of the 0.062 mg/mL Standard to wells E-1 through E-3
6. Add 25 µL of dH2O to wells F1 through F3
Adding the “unknown” Samples 1.
Add 25 µL of each protein sample dilution to the corresponding wells
| 1 | 2 | 3 | 4 | 5 | 6 |
A | 1.0 mg/ml | 1.0 mg/ml | 1.0 mg/ml | Sample 1:4 | sample 1:4 | sample 1:4 |
B | 0.5 mg/ml | 0.5 mg/ml | 0.5 mg/ml | sample 1:16 | sample 1:16 | sample 1:16 |
C | 0.25 mg/ml | 0.25 mg/ml | 0.25 mg/ml | sample 1:64 | sample 1:64 | sample 1:64 |
D | 0.125 mg/ml | 0.125 mg/ml | 0.125 mg/ml | Sample 1:256 | Sample 1:256 | Sample 1:256 |
E | 0.062 mg/ml | 0.062 mg/ml | 0.062 mg/ml | Sample 1:1024 | Sample 1:1024 | Sample 1:1024 |
F | 0 mg/ml | 0 mg/ml | 0 mg/ml | | | |
Hands on activity
Instructions
Adding the Detection Reagent (Bradford)
1. Using the (P200) and tips, add 200 µL of the Bradford reagent to each well. Once you start, it should be done quickly so that one sample is not exposed to the reagent significantly longer than another. Start at the highest dilution (“0” for the Standards) and add the reagent as you go up in concentration, touching only the side of the well with your pipet tip. Repeat with each sample, starting at the 1:1024 dilution and moving up. Notice that each standard and sample is done in triplicate.
2. Incubate on your table for 10 minutes
3. Bring your plate to the Spectrophotometer (Plate Reader) to measure the absorbance of each well (Wavelength will be set between 540 and 590 nm) 4. If you do not have a spectrophotometer or Plate Reader, visually compare the color blue of your samples to the standards. The standard color that most matches the “unknown” is an estimate of the protein concentration. Remember to multiply by the dilution factor (2,4,10, 100, or 1000) to get your final result.
Assessment
The students will look at the colors ro determine the concentration of the protein.
If you have a spectrophotometer, I would have them use that to check their work of their sample concentration.
I would have them do a lab write-up for this lab for the assessment. rubric posted to the right.
Differentiation
One way to differentiate in this lesson is to provide chunking of the lesson and or also work the lab with them. You can go through the procedures together.
You can also put them with students that know what to do and can help them along.
Remediation
Extension/Enrichment
Students who are successful right away can create more ways to test proteins.
The students can also teach other groups and help them.