1. EXPERIMENTS 2.1-2.4: CELL
FRACTIONATION, PROTEIN ASSAY,
SDH ASSAY AND
SDS-PAGE
Michael Truck - Biology 104 - TA: Elizabeth Braschayko

2. PURPOSE
Cell Fractionation - To Understand the laboratory
technique that uses differential centrifugation to
separate the different components of the cell,
resulting in nuclear, mitochondrial, microsomal, and
soluble fractions.
Protein Assay - To determine the protein
concentration of each fraction obtained.
Succinate Dehydrogenase (SDH) Assay – Used to
test the enzymatic activity of SDH in the different
cellular fractions.
SDS-PAGE - to identify the unknown proteins in
the mitochondrial proteome

3. EXPECTED RESULTS
Mitochondrial fractions should have highest SDH
specific activity (electron transport)
Homogenate fraction should have highest protein
concentration but lower SDH activity than
mitochondria (contains unbroken cells + mixture of
organelles)
Soluble fraction should have lowest protein
concentration and lowest SDH specific activity
(smallest cellular particles, lowest amount of
proteins)

4. SUMMARY OF PROCEDURE
Cell Fractionation: homogenized beef liver, added some
buffer, centrifuged at different speeds and intervals to
get the different cellular fractions.
Protein Assay: used Bovine Serum Albumin (BSA) to make
standard curve using known BSA concentrations; made
dilutions of cell fractions and calculated protein
concentrations using linear equation of standard curve

5. SDH Assay: prepared dilutions of each fraction, added
enzyme mixture and then placed in 37°C water bath;
read absorbance at t=0 min., t=3 min. and t=15 min.;
We then used this data with the protein concentrations
to calculate the specific activity of SDH in each sample
SDS-PAGE: Added acetone to protein sample. Incubated
for 40 mins., centrifuged, disposed of the supernatant,
dried the pellet. Added 8ug buffer and 10ul of 2x gel
loading dye and placed in water bath. Then loaded onto
SDS-PAGE gel.

6. RESULTS – BSA STANDARD CURVE
Tube Dilution Factor BSA Volume (mL) ddw Volume (mL) [BSA]ug/mL Conc. BSA Amount in Assay (ug) Abs. @ 595
1 0 1 0 1000 100 0.256
2 4/5 0.8 0.2 800 80 0.252
3 3/5 0.6 0.4 600 60 0.241
4 2/5 0.4 0.6 400 40 0.235
5 1/5 0.2 0.8 200 20 0.220
6 1/10 0.1 0.9 100 10 0.202
7 - - 1.0 0 0 0.0
8 - - - 0 0 0.079

7. RESULTS – CELL FRACTIONS
Tube Dilution Factor Volume (mL) Fr. + d.w. ABS average Conc. (mg/mL)
H1 1/10 0.5+4.5 0.234 2.22
H2 1/20 0.5+9.5 0.064
N1 1/5 0.5+2.0 0.286 3.21
N2 1/10 0.5+4.5 0.143
M1 1/5 0.5+2.0 0.386 7.02
M2 1/10 0.5+4.5 0.241
S1 1/5 0.5+2.0 0.342 5.35
S2 1/10 0.5+4.5 0.188

8. RESULTS – SDH ASSAY
Tube t=0 t=3 t=15
1 Blank - - -
2 H 0.994 0.373 0.256
3 N 0.922 0.318 0.197
4 M 1:5 0.952 0.383 0.286
5 M 1:10 0.910 0.334 0.230
6 M 1:25 0.894 0.332 0.265
7 S 0.930 0.480 0.298
Tube SDH Specific Activity
2( Homogenate, 1:2) 141
3(Nuclear, 1:2) 124
4(Mitochondrial, 1:5) 34.6
5(Mitochondrial, 1:10) 44.2
6(Mitochondrial, 1:25) 26.8
7(Soluble, 1:2) 74.3

9. 0
25
50
75
100
125
150
2( Homogenate, 1:2)
3(Nuclear, 1:2)
4(Mitochondrial, 1:5)
SDH Assay
5(Mitochondrial, 1:10)
6(Mitochondrial, 1:25)
SDH SPECIFIC ACTIVITY
7(Soluble, 1:2)

10. DISCUSSION
Here we can see that the Homogenate fraction
showed the highest enzymatic activity and the
mitochondrial fractions showed the lowest
activity. This also shows that the 1:10 dilution of
mitochondria is higher than the 1:5 dilution
which is wrong. This data is all contradictory as
to the correct outcome of what the results
should be. Some sources of error could be that
the mitochondria was not fully broken open in
the homogenization step. This being said, SDH
could have not been interacting with the DCPIP
due to the fact that the mitochondrial wall was
not broken open.

11. MORE SOURCES OF ERROR
the fractionation process was not carried
out long enough for the cells to
fractionate.
The absorbance readings contained Error
from the spec-20 machine having dirty
tubes or from malfunction of the machine
itself
Human error (vague - covers many things)

12. DOES ANY 1
HAVE
QUESTIONS
?