Lab Sheet
Got Protein?
Objectives:

Perform a serial dilution

Use a spectrophotometer to quantify protein concentrations

Use a standard curve to determine concentrations of unknown solutions
IMPORTANT: Read the background and procedure then answer the pre-lab questions before performing the experiment.
Background:
Proteins are essential to maintaining a healthy metabolism. Many proteins are enzymes that control the rate of chemical reactions involved in digestion; while others act as hormones that signal cells as part of normal growth and immune response. Proteins are also one of the many building blocks that the body uses to make muscles, skin and other tissues. Because humans and other animals cannot make all the basic units of proteins (amino acids), proteins are also an important part of our diet. Many people use protein shakes and protein supplements to try to lose weight, build muscle mass or perform better in sports. But how can you be sure that a protein shake actually contains as much protein as it claims?
Proteins themselves are typically colorless and can be difficult to detect without a specific indicator. Biuret reagent changes from blue to violet when it reacts with proteins, allowing you to qualitatively conclude that protein is present in a solution. Biuret reagent can also be used quantify the amount of protein in a sample. A concentrated protein solution will give a strong biuret reaction and turn intensely purple. In contrast, a dilute solution will have a weak reaction with biuret reagent and turn a pale violet. A machine called a spectrophotometer can be used to determine the specific amount of protein present by measuring the amount of light absorbed by the stained proteins.
In this experiment, you will perform a serial dilution of bovine serum albumin (BSA) protein to produce solutions with a range of known concentrations. You will then do a biuret assay on these protein standards, and measure the amount of light absorbed using a spectrophotometer. Next, you will create a standard curve—a graph of the amount of light absorbed by each standard vs. the concentration of each standard. The standard curve can then be used to determine the protein concentration of several unknown solutions.
Materials (per group):
5 15 ml empty test tubes
2 5ml Pipettes with bulb
1 ml pipette
BSA stock solution (40,000 µg/ml)
Distilled water
Biuret reagent
Test tube rack
Marking pen

Lab Sheet
Beaker or cup for waste
2 Unknown solutions
5 cuvettes or spectrophotometer tubes
1 spectrophotometer
Stop watch/ timer
Lab wipes
Classroom vortex (optional)
Procedure:
IMPORTANT: Turn on the spectrophotometer before preparing the protein standards.
The instrument must be warm for at least 15 minutes before use.
Preparation of Protein Standards: Refer to Figure 1 Steps to Perform a Serial Dilution
1.
Label 5 empty test tubes 1 through 5.
2.
Using a 5 ml pipette, place 5ml of distilled water into each tube labeled 2-5. Then pipette
9.5 ml of water into the tube labeled 1. Reminders: You may use the same pipette to put the water into all the tubes.
3.
Using a 1 ml pipette, add 0.5 ml of the BSA stock solution into tube 1. Pipette the solution up and down several times to mix well.
You may find it helpful to refer to
Figure 1 Steps to perform a serial dilution.
4.
Using a 5 ml pipette, remove 5 ml of the BSA solution from tube 1 and add it to the water in tube 2. Pipette the solution up and down several times to mix well.
5.
Using the same 5 ml pipette, remove 5 ml of the BSA solution from tube 2 and add it to the water in tube 3. Pipette the solution up and down several times to mix well.
6.
Using the same 5 ml pipette, remove 5 ml of the BSA solution from tube 3 and add it to the water in tube 4. Pipette the solution up and down several times to mix well.
7.
Using the same 5 ml pipette, remove 5 ml of the BSA solution from tube 4 and
DISCARD in waste container or the sink.
8.
Add nothing to tube 1. All 5 tubes should now contain an equal volume of 5 ml.
Figure 1. Steps to perform a serial dilution
Stock
0.5 ml
9.5 ml
H
2
O
Concentration
1
5 ml
5 ml H
2
O
2
5 ml
5 ml H
2
O
3
5 ml
5 ml H
2
O
4
5 ml
5 ml H
2
O
DISCARD
5

Lab Sheet
Performing the Biuret Assay
1.
Using a clean 5 ml pipette, add 2.5 ml of biuret reagent to each of the tubes 1-5. Do NOT touch the BSA solution with the pipette. Reminder: If you avoid touching the BSA solution with the pipette, you can use the same pipette to add biuret reagent to each tube.
2.
Add 2.5 ml of biuret reagent to each of your unknown tubes.
3.
Mix the solutions thoroughly by vortexing or by rotating the tubes between the palms of your hands as demonstrated by your instructor.
4.
Allow the tubes to stand for at least 10 minutes to allow the color to develop fully.
Reminder: Biuret reagent changes from blue to violet when it comes in contact with a protein. This color change will remain stable for up to one hour after the solutions are mixed.
5.
While you are waiting for the color to develop, prepare the spectrophotometer according to your instructor’s directions. a.
The instrument must have been warm for at least 15 minutes before using it. b.
Set the wavelength to 540 nm. c.
With the chamber empty, adjust the meter to read “100” on the % transmittance scale.
 This step is referred to as “blanking the spectrophotometer.” Reminder:
Always close the cover of the sample chamber. d.
Then “blank” the spectrophotometer using the contents of tube 5 (biuret reagent + water). Transfer ___ ml of the contents of tube 5 into a clean cuvette or spectrophotometer tube using a clean pipette. Wipe the tube (or cuvette) with a lab wipe and place it into the sample chamber. Close the cover and set the meter to “0” on the absorbance scale.
6.
After blanking the instrument, read the absorbance (A) for each protein standard tubes 1-
4 and the unknown samples. a.
Transfer 1 ml of the contents of each tube into a clean cuvette or spectrophotometer tube using a clean pipette. b.
Wipe off the sample, insert it into the chamber and close the cover before reading the absorbance . c.
Record your results in Table 1 Protein Concentrations and Absorbance at 540 nm on the Student Worksheet.

Reminder: Check the spectrophotometer after removing each sample.
Readjust the “zero” and “reblank” if necessary before examining the next sample.
Extension:
If time allows, students will investigate absorption spectrum of biuret reagent and/or the protein content in protein drinks.

Student Worksheet
Name: ___________________________________________________ Date: ______________
Got Protein?
IMPORTANT: Read the background and procedure then answer the Pre-Lab questions before performing the experiment.
Pre-Lab Questions:
1. Describe two functions of proteins in the human body.
2. What is the difference between qualitative and quantitative data?
3. Make a prediction about the effect protein concentration will have on the amount of light absorbed by a sample.
Observations and Results:
1. The simple equation that follows will allow you to calculate the concentration of BSA in each of your standard solutions.
C
1
V
1
=C
2
V
2
C
1
is the concentration of the original solution
C
2
is the concentration of the final solution in the tube
V
1
is the volume of the amount transferred into the tube
V
2
is the volume of the solution in the tube before you remove any BSA
Example: Tube 1 C
1
= 40,000 µg/ml BSA Stock Solution
C
2
= ?
V
1
= 0.5 ml ( V
1
=0.5 ml for tube 1 only; V
1
=5 ml for tubes 2-5 )
V
2
= 10 ml
(40,000 µg/ml)(0.5 ml) = (C
2
)(10 ml)
20,000 µg = (C
2
)(10 ml)
2,000µg/ml = C
2
For tubes 1-5 you already know C
1
, V
1
and V
2
. All you need to do is plug in the numbers and

Student Worksheet solve for C
2
. Record these results in the "BSA Concentration" column of Table 1 Protein
Concentration and Absorbance at 540 nm.
Table 1. Protein Concentration and Absorbance at 540 nm
Test Tube Number BSA Concentration
(µg/ml)
A
540
1
Observations
2
3
4
5 (blank)
Unknown __
Unknown __
2. Plot your data on the graph below to create a standard curve (BSA concentration vs.
Absorbance at 540 nm). Reminder: label your x and y-axis and title your graph.

Student Worksheet
Post-Lab Analysis:
1. Using your standard curve, determine the concentrations of your unknown solutions. Record the results in Table 1 Protein Concentration and Absorbance at 540 nm.
2. Imagine you were explaining this experiment to a friend and they said that you should have used water (without biuret reagent) to blank the spectrophotometer. How would you respond?
Hint: Think about zeroing a balance when you are using a weighing boat.
3. Could you use your standard curve to determine the concentration of starch in an unknown solution? Why or why not?
4. Was your prediction about the effect protein concentration would have on the amount of light absorbed by a sample accurate? Why or why not?
5. Write a paragraph describing what you learned in this lab. This paragraph should include the purpose of this lab, a summary of results and the significance of the results. Some of the questions above will get you started.