The effect of different diets on glucose and hexokinase IV in rat liver
Exercise 1 Protein assay
Bradford protein assay
You are now going to determine protein concentration of your liver samples using the Bradford assay. The challenge here is that you will need to dilute your samples enough so that the readings lie within the linear range of the protein standard curve.
Methodology
Set up your protein standard dilutions in 7 Eppendorf tubes. Six serial 1:2 dilutions should be sufficient as that will provide a range of 0.1 mg/mL – 0.1 mg/mL. You can set these seven dilutions in Eppendorf tubes and label them SN, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64.
Pipette 500 mL of tissue buffer into tubes 1:2, 1:4, 1:8, 1:16, 1:32, 1:64.
For your neat standard (SN), add 500 mL of your protein standard to an Eppendorf tube.
For the first dilution (1:2), add 500 mL of your protein standard to an Eppendorf tube. Mix with the pipette.
Then continue to do serial dilutions for the remainder of the dilutions (1:4, 1:8, 1:16, 1:32, 1:64) as follows:
Add 500 mL of your 1:2 dilution to the 1:4 tube, mix with the pipette.
Add 500 mL of your 1:4 dilution to the 1:8 tube, mix with the pipette.
Add 500 mL of this 1:8 dilution to the 1:16 tube, mix with the pipette.
Add 500 mL of this 1:16 dilution to the 1:32 tube, mix with the pipette.
Add 500 mL of this 1:32 dilution to the 1:64 tube, mix with the pipette.
You should now dilute your tissue samples.
You will need the following dilutions: 1:10, 1:100, 1:1000. (Remember: your sample is already 1:10 from Step 7b Exercise 1).
Add 900 mL Tissue Buffer to the 1:100 and 1:1000 Eppendorf tubes.
Transfer 100 mL of your liver sample to the 1:100 tube, mix with the pipette.
Transfer 100 mL of your 1:100 dilution to the 1:1000 tube, mix with the pipette.
Transfer 150 mL of your standard curve, unknown dilutions and blank (tissue buffer only) into the 96 well plate as outlined in the table on the next page.
1 2 3 4 5 6 7 8 9 10 11 12
A SN 1:2 1:4 1:8 1:16 1:32 1:64 Sample 1:10 Sample 1:100 Sample 1:1000 Tissue buffer
B SN 1:2 1:4 1:8 1:16 1:32 1:64 Sample 1:10 Sample 1:100 Sample 1:1000 Tissue buffer
C SN 1:2 1:4 1:8 1:16 1:32 1:64 Sample 1:10 Sample 1:100 Sample 1:1000 Tissue buffer
D
E
F
G
H
Add 150 µL of the Coomassie dye solution to each well. You can use the multichannel pipette if needed.
Read your 96 well plate in the micro-plate reader at a wavelength of 595 nm.
Plot your absorbances as a Standard Curve using Excel.
In Excel, using the equation of your Standard Curve and dilution factors, calculate the original protein concentration of your liver sample.
Tube:
Dilution Conc. of
BSA
mg/mL
A540
(Reading 1)
A540
(Reading 2)
A540
(Reading 3)
Average A540
0 (blank)
Blank Blank Blank
SN
1:2
1:4
1:8
1:16
1:32
1:64
Exercise 2 Hexokinase IV specific activity
Calculate the specific activity of hexokinase IV in the original (undiluted) hexokinase suspension using the table below to guide you:
Calculation Steps Units Sample Dilution
1 in 100
(Tubes A, B & C)
a. A540 – A:
b. A540 – B:
c. A540 – C:
d. Average A540 –
e. Concentration of reducing sugar (from standard curve) mmol/mL
f. Rate of Reaction (e/5 min) mmol/
(mL.min)
g. Adjust for Dilution factors
(f X dilution factor) mmol/
(mL.min)
h. Amount glucose broken down (one µmole of glucose produces 1 µmoles reducing sugar) (g/1) mmol/
(mL.min)
i. Units of Hexokinase IV activity per mL (same value as h) U/mL
(where 1 U =
1 mmol/min)
j. Protein concentration of your liver sample (from Practical 8 Exercise 1) mg/ml
k. Specific activity of Hexokinase IV (i / j) U/mg or umol/min/mg
