Francis Boyer

Biol. 3204 Lab

Partners: Raye-Julianne Delos-Reyes, Taylor Campbell

December 1st, 2023

Sec. 15

Cell Fractionation

Objective

The objective of this experiment was to use repeated differential centrifugation on

beef liver homogenate to yield cellular fractions from disrupted cell membranes, and then assay

the cellular contents of these fractions using enzyme assays and fluorescence microscopy. The

relative enrichment of the fractions was determined via fluorescence microscopy, the protein

content via Bradford assay, and the fraction contents represented by enzyme activity via

enzyme assays. The results of these assays allowed for the determination of the cellular

components found within the fractions.

Methods

Homogenizing Liver Tissue

Fresh beef liver was minced and combined with 10ml of ice-cold homogenizing medium,

noting 5ml of displacement volume. Additional homogenizing medium was added to bring the

mixtures total volume to 5 times the livers displacement volume; 25ml. This mixture was

processed into 23ml of homogenate, and an equal volume of homogenizing medium was

added.

Differential Centrifugation

The remaining homogenate solution was centrifuged at 1000 x g for 2 minutes. The

yielded supernatant was centrifuged at 15,000 x g for 20 minutes, yielding “Supernatant 2” and

“Pellet 2.” Pellet 2 was resuspended in 5ml of homogenizing fluid. The pellet yielded from
centrifugation of the homogenate solution was resuspended in 2ml of homogenizing fluid and

centrifuged at 300 x g for 5 minutes. The yielded supernatant was “Pellet 1 Resuspended.”

Analysis of Protein concentration via Bradford Assay

The protein content of the fractions was determined via Bradford Assay (Bradford, 1976).

Analysis of Fraction Content via Fluorescence Microscopy

Samples of the liver fractions on slides were dried and combined with 50µl of fluorescent

dye mixture and incubated for 10 minutes at room temperature, before being rinsed with water.

Two drops of 50% glycerol mounting solution was added, and the fractions were examined.

Analysis of Fraction Content via Enzyme Assay - Succinate Dehydrogenase

Nine solutions of 0.2ml of phosphate buffer, pH 7.4, 0.1ml of nitroblue tetrazolium (NBT),

0.1ml of 1% Triton WR-1339, and 0.1ml of succinate were prepared. To four of these solutions,

0.5ml of each fraction was added, respectively. To four more of these solutions, 0.45ml of

phosphate buffer, pH 7.4 was added, as well as 50µl of each fraction, respectively. To the

remaining solution, 0.5ml of phosphate buffer was added. These solutions were incubated at

37°C for 30 minutes, before 2ml of 2% SDS was added to each to stop the reactions. The

absorbance of these solutions was then measured at 630nm.

Analysis of Fraction Content via Enzyme Assay - Acid Phosphatase

Nine solutions of 100µl glycine buffer and 50µl of PNPP were prepared. To four of these

solutions, 100µl of each fraction was added, respectively. To four more of these solutions, 90µl

of phosphate buffer was added as well as 10µl of each fraction, respectively. To the remaining

solution, 100µl of phosphate buffer was added. These solutions were incubated at 37°C for 30

minutes, before 2.75ml of ice-cold 0.2M Na3PO4, pH 12 was added to each to stop the

reactions. The absorbance of these solutions was then measured at 410nm.

Results

Table 1: BSA Standards Results

Assay Tube Desired Conc. Volume of 1 mg/ Vol. of phosp. Absorbance at

Number Of BSA (mg/ml) ml BSA (µl) buffer (µl) 595nm
1 = Blank 0 0 60 0

2 0.2 12 48 0.091

3 0.4 24 36 0.193

4 0.6 36 24 0.309

5 0.8 48 12 0.420

6 1 60 0 0.476

Figure 1: Chart of Absorbance at 595nm vs Desired Concentration of BSA.

(Data Absorbance at 595nm and Desired Concentration of BSA (mg/ml) courtesy of Bench 3)
Table 2.1: Protein Content of Cellular Fractions

Cellular Dilution of Absorbance at Protein (mg/ml)

Volume (ml)
Fraction Fraction 595nm from Standards
Homogenate 23 5x 0.5 1.01

Pellet 1 (Resus.) 1.5 5x 0.145 0.29

Pellet 2 5.1 5x 0.440 0.89

Supernatant 2 32 5x 0.480 0.97

Table 2.2: Protein Content of Cellular Fractions cont.

% of Homogenate
Cellular Fraction Protein (mg/ml)* Total Protein (mg)
protein
Homogenate 5.05 116.15 100

Pellet 1 (Resusp.) 1.45 2.18 1.88

Pellet 2 4.45 22.70 19.54

Supernatant 2 4.85 155.20 133.62

(*after taking dilutions into account)

Table 3: Composition of Cellular Fractions by Fluorescence Microscopy

Sample DAPI Mitotracker

Homogenate 10 9

Pellet 1 (Resusp.) 7 7

Pellet 2 5 8

Supernatant 2 2 2

Calculations

(Figure 1 & Table 2.1) Proteins (mg/ml) from Standard: 0.5 = 0.4976x - 0.0006 = 1.01

(Table 2.2) Total (mg) Protein: Volumes (ml) × Protein (mg/ml) = 23 × 5.05 = 116.15

(Table 2.2) % of Homog. Protein: Total Protein (mg) / Total Protein of Homog. (mg) × 100% =

(2.18 / 116.15) × 100% = 1.88%

Table 4: Activity of Succinate Dehydrogenase in Cellular Fractions

Total Volume of Absorbance at

Cellular Fraction Activity/ml
Fraction Used (ml) 630nm
Homogenate 0.50 / 0.05 0.181 / 0.013 0.362 / 0.26

Pellet 1 (Resusp.) 0.50 / 0.05 0.014 / 0.005 0.028 / 0.10

Pellet 2 0.50 / 0.05 0.082 / 0.008 0.164 / 0.16

Supernatant 2 0.50 / 0.05 0.043 / 0.003 0.086 / 0.06

Table 5: Activity of Acid Phosphatase in Cellular Fractions

Total Volume of Absorbance at

Cellular Fraction Activity/ml
Fraction Used (ml) 410nm
Homogenate 0.10 / 0.01 1.000 / 0.229 10.00 / 22.90

Pellet 1 (Resusp.) 0.10 / 0.01 0.145 / 0.152 1.45 / 15.20

Pellet 2 0.10 / 0.01 0.830 / 0.182 8.30 / 18.20

Supernatant 2 0.10 / 0.01 0.640 / 0.185 6.40 / 18.50

Table 6.1: General Table of Enzyme Activities - Succinate Dehydrogenase

Cellular Total Volume Protein Spec.Activity

Activity/ml Total Activity
Fraction (ml) (mg/ml) (Activity/mg)
Homogenate 0.362 / 0.26 23 8.326 / 5.98 5.05 0.072 / 0.051

Pellet 1 (RS.) 0.028 / 0.10 1.5 0.042 / 0.15 1.45 0.019 / 0.069

Pellet 2 0.164 / 0.16 5.1 0.836 / 0.816 4.45 0.037 / 0.036

Supernat. 2 0.086 / 0.06 32 2.752 / 1.92 4.85 0.018 / 0.012

Table 6.2: General Table of Enzyme Activities - Acid Phosphatase

Cellular Total Volume Protein Spec.Activity

Activity/ml Total Activity
Fraction (ml) (mg/ml) (Activity/mg)
Homogenate 10.00 / 22.90 23 230 / 526.7 5.05 1.980 / 4.535

Pellet 1 (RS.) 1.45 / 15.20 1.5 2.175 / 22.8 1.45 1.00 / 10.483

Pellet 2 8.30 / 18.20 5.1 42.33 / 92.82 4.45 1.865 / 4.089

Supernat. 2 6.40 / 18.50 32 204.8 / 592 4.85 1.320 / 3.814

Calculations cont.

(Table 4 & 5) Activity/ml: Absorbance / Total Vol. of Fraction Used (ml) = 0.181 / 0.50 = 0.362

= 0.013 / 0.05 = 0.26

(Table 6.1 & 6.2) Total Activity: Total Vol. (ml) × Activity/ml = 0.362 × 23 = 8.326

= 0.26 × 23 = 5.98

(Table 6.1 & 6.2) Specific Activity: Activity/ml / Protein (mg/ml) = 0.362 / 5.05 = 0.07

= 0.26 / 5.05 = 0.05

Discussion

[Discussion goes here]

References

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protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248.

Karp, G. et al. 2019. Cell and Molecular Biology, 9th ed. Wiley.

Ninfa, A. J., and Ballou, D. P. 1998. Fundamental Laboratory Approaches for Biochemistry and

Biotechnology. Fitzgerald Science Press (Bethesda, MD).

Padh, H. 1992. Organelle isolation and marker enzyme assay. Pages 129-146, in Tested studies

for laboratory teaching, volume 13, Editor C. A. Goldman. Proceedings of the 13th

Workshop/Conference of the Association for Biology Laboratory Education.

Sedmark, J. J., and S. E. Grossberg. 1977. A rapid, sensitive and versatile assay for protein

using Coomassie Brilliant Blue G250. Anal. Biochem. 79: 544.

Sheffield, J. 2023. Biology 3204 Lab Manual. Temple University.