1
LSC 355
Cell Biology Laboratory
Name: ___________________________________
Datum Sheet for Lab 9
Analysis of Proteins in Different Animal Organs,
Part 1
B.
C.
Preparation of Acetone Powder Suspensions
1.
Which organ acetone powder did you use? ____________________
2.
How many mg of powder did you use?
3.
About how many ml of supernatant did you obtain? ______________
____________________
Protein Concentrations of Acetone Powder Suspensions
1.
tube
Fill in the following chart with the data for the BSA standard curve.
volume of
water
volume of
BSA
g of
BSA
A595
average ABS595
1
50 l
0 l
__________
_________
_________
2
45
5
__________
_________
_________
3
45
5
__________
_________
4
40
10
__________
_________
5
45
10
__________
_________
6
35
15
__________
_________
7
35
15
__________
_________
8
30
20
__________
_________
9
30
20
__________
_________
10
20
30
__________
_________
_________
_________
_________
_________
2
11
20
30
__________
_________
12
10
40
__________
_________
13
10
40
__________
_________
14
0
50
__________
_________
15
0
50
__________
_________
_________
_________
2.
Attach to this data sheet your graph of the BSA standard curve.
3.
Using your standard curve, determine the conversion factor for relating
absorbance at 595 nm to g of protein and provide it in the space below.
____________
4.
Fill in the following chart with the data for the protein assay of the acetone
powder suspension. Calculate g amount using conversion factor in #3 for those
tubes that are in the range of your standard curve.
tube
extract
water
extract
A595
g
1
-----
50 l
0 l
_________
_______
2
stock solution
45
5
_________
_______
3
40
10
_________
_______
4
30
20
_________
_______
5
0
50
_________
_______
45
5
_________
_______
7
40
10
_________
_______
8
30
20
_________
_______
9
0
50
_________
_______
10
1/100 dilution 45
5
_________
_______
11
90
10
_________
_______
6
1/10 dilution
3
12
30
20
_________
_______
13
0
50
_________
_______
5.
Choose three representative tubes from above and calculate the concentration of protein
extract in mg/ml based on that tube. Indicate the tubes you have chosen and show your
calculations
Tube #
g
______
______
______
______
______
______
______
______
______
Calculations
mg/ml
6.
Using the three representative tubes above, give the average value of the protein
concentration of your acetone powder suspension in mg/ml and show your calculations.
7.
Using the three representative tubes above, give the standard error of the protein
concentration of your acetone powder suspension in mg/ml and show your calculations.
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Data Sheet for Lab 9
Analysis of Proteins in Different Animal Organs
Part 2
A.
Preparation of Protein Samples
1.
2.
Give the name of the tissue extract and the protein concentration that you
determined last week.
Tissue Extract:
__________________
Protein Concentration (mg/ml):
__________________
Give the calculated dilution factor you used to make a 2 mg/ml solution.
________________
3.
C.
Give the amount of extract and the amount of water you used to prepare 300 μl
of a working solution at 2 mg/ml.
extract:
_______________
water:
_______________
Analysis of Standard Protein Gels
1.
Attach to this data sheet the printed digital image of the SDS-polyacrylamide gel.
Be sure that you label the lanes number the bands in your gel images. Remember
each person needs to do this not just one for the group.
2.
Fill in the following chart to indicate the mobilities in (cm) of each of the standard
proteins in the SDS 7-L and SDS 6-H mixtures and the purified proteins on the
gel that was made by your group. Measure the distance in cm from the bottom of
each well to the center of each band. Use NV to indicate that a particular protein
band was not visible.
purified proteins:
protein
molecular mass
mobility (cm)
gel A gel B
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rabbit glyceraldehyde-3-phosphate
dehydrogenase
36,000
___________
bovine albumin
66,000
___________
phosphorylase b
97,000
___________
protein
molecular mass
mobility (cm)
gel A gel B
bovine alpha-lactalbumin
14,200
___________
soybean trypsin inhibitor
20,100
___________
bovine trypsinogen
24,000
___________
bovine carbonic anhydrase
29,000
___________
rabbit glyceraldehyde-3-phosphate
dehydrogenase
36,000
___________
egg albumin
45,000
___________
bovine albumin
66,000
___________
protein
molecular mass
mobility (cm)
gel A gel B
bovine carbonic anhydrase
29,000
___________
egg albumin
45,000
___________
bovine albumin
66,000
___________
rabbit phosphorylase b
97,400
___________
E. coli beta-galactosidase
116,000
___________
rabbit myosin
205,000
___________
SDS-7L:
SDS-6H:
3.
Attach to this data sheet the standard curve for the proteins in the 7-L and 6-H
mixtures for the 8-16% polyacrylamide gels. Plot mobility (in cm) on the X axis
and log molecular weight on the Y axis. Include the data from both the 7-L
6
and 6-H mixtures of standard proteins and both gels on a single graph.
Draw the best fit line you can through the data points.
4.
You are provided a protein that has a quaternary structure composed of three
subunits. The molecule mass of each of the three subunits is as follows:
Subunit A
17,000 daltons
Subunit B
25,000 daltons
Subunit C
30,000 daltons
If this protein was fractionated on a SDS-PAGE gel like you did in class, what would you expect
in the lane that contained this protein? Be specific, include number and size of band and also the
expected distance migrated based on your standard curve from #3.