UST College of Science

Analytical Chemistry 1 Lab (CHM414L)

Experiment No. 1
Pipetting: Determine the protein concentration in a corn solution

Techniques in the Lab:

 Pipetting using a micropipettor
 Serial Dilution
 Bradford Assay

I. Objectives:
 To prepare different solution concentrations using a micropipettor and serial dilution
 To determine the protein (amino acid) concentration in a genetically modifed corn solution using the Bradford
assay

II. Materials:
 Phosphate buffer
 Bradford reagent
 Bovine Serum Albumin (BSA)
 Corn (Zea mays)
 Micropipettors
 Spectrophotometer (Microplate Reader)

III. Procedure:
1. Obtain a sample of corn. (Zea mays)
2. Grind 100.0 g of corn sample with 10.00 mL of phosphate buffer using a mortar and pestle.
3. Filter the mixture and obtain the filtrate. Discard the residue.
4. Dilute the filtrate using a serial dilution. (Fill-up the table based on what you did in the simulation.)
Concentration Volume of solution Volume of buffer
1:5 uL of sample 800 µL
1:50 900 µL
1:500
1:5000

5. Prepare five standard solutions of 0.05, 0.10, 0.20, 0.30, 0.40, and 0.50 mg/mL of BSA in phosphate buffer.
6. Obtain 10 µL of each solution and transfer it into a well of a microplate following this arrangement:
A 1 (Sample - Trial 1) 2 (Sample -Trial 2) 3 (Standard – Trial 1) 4 (Standard – Trial 2)
B 1:5 1:5 0.50 mg BSA / mL 0.50 mg BSA / mL
C 1:50 1:50 0.40 0.40
D 1:500 1:500 0.30 0.30
E 1:5000 1:5000 0.20 0.20
F 0.10 0.10
G 0.05 0.05
7. Set the spectrophotometer (microplate reader) to 595 nmn.
8. Place the microplate inside the instrument.
9. Start the measurement and record the absorbance of each solution.
UST College of Science
Analytical Chemistry 1 Lab (CHM414L)

IV. Results:

Sample Absorbance Absorbance Average Standard Absorbance Absorbance Average

Solution – Trial 1 – Trial 1 Absorbance Solution – Trial 1 – Trial 1 Absorbance
(Sample) (mg/mL (Standard)
BSA)
1:5 0.50
1:50 0.40
1:500 0.30
1:5000 0.20
0.10
0.05

V. Treatment of results
1. Get the average of each sample and standard solution.
2. Graph and perform a linear regression using the standard solution concentration (x) and average absorbance of
standards (y) to prepare a calibration curve. Determine the equation of the line.
3. Calculate the concentration of protein (in terms of BSA) of the sample solution within the calibration curve using the
equation of the line.
4. Calculate the amount of protein found in the corn sample in terms of mg/g of corn.