BIO 220 Laboratory Exercises
Laboratory I: Subcellular Fractionation and
Protein Determination of Subcellular Compartments
The goal of this session is to determine the relative distribution of protein within rat liver cells.
This exercise consists of three parts:
1) Mechanical disruption of rat liver cells - production of the Homogenate (H)
2) Differential centrifugation - collection of three subcellular fractions:
Nuclear Pellet (NP), Mitochondrial Pellet (MP), and Post-Mitochondrial Supernatant (PMS)
3) Quantitation of protein in each fraction
Introduction
Many studies in cell physiology and biochemistry require relatively pure preparations of
subcellular organelles. General purification procedures require that the cells first be disrupted
mechanically, using a tissue grinder or a Waring blender, before separation of the organelles.
Organelles are separated from each other using a technique called differential centrifugation (see
Fig. 1). Differential centrifugation is a stepwise sedimentation of successively smaller particles.
The force exerted on the particles is expressed as a relative centrifugal force (RCF) in terms of
gravitational units (x g). The RCF is a function of the speed of centrifugation in revolutions per
minute (RPM), and the distance of a particle from the center of rotation (radius). Knowing the
RPM and the radius of the centrifuge head, one can readily determine the RCF.
The rate at which a particle travels in a centrifugal field is a function of its size, shape and
density. Large structures, like the nucleus, will sediment when a relatively low centrifugal force
is applied (600 x g for 5 min). The nuclear fraction contains nuclei, intact cells and cell debris.
For further separation, the supernatant is transferred to another centrifuge tube and spun for 20
min at 10 000 x g. The sedimented material is the mitochondrial fraction. Lysosomes,
peroxisomes, endoplasmic reticulum and ribosomes are found in the post-mitochondrial
supernatant.
In subcellular fractionation experiments, researchers commonly express concentrations or
activities of compounds with respect to protein content in that fraction. This convention is
recommended because tissue disruption and centrifugation techniques are not quantitative, with
loss of material at each step of the procedure. Therefore, the protein concentration (mg/mL) and
total protein (mg) must be determined for each subcellular fraction: homogenate (H), nuclear
pellet (NP), mitochondrial pellet (MP), and post-mitochondrial supernatant (PMS). This
information can be used to calculate the protein distribution within the cell.
SAVE ALL FRACTIONS FOR FUTURE EXPERIMENTS. THE MITOCHONDIRAL
FRACTION WILL BE USED FOR THE NEXT EXPERIMENT IN ENZYME KINETICS.
Figure 1. Scheme for fractionation of cells by differential centrifugation. This is Figure 5-23 from Molecular Cell
Biology, 4th edition1, reproduced with notations. Centrifugation conditions in this lab may differ.
Procedure
Part 1) Mechanical disruption of rat liver cells - production of the Homogenate (H):
Safety Notice:
The Omni-mixer is a potentially dangerous instrument if used improperly. Do not operate the
Omni-mixer without supervision of the instructor.
Following lethal administration of sodium pentobarbital to an adult rat, the liver is
removed and weighed. The intact liver is placed in a beaker containing ice-cold 0.25 M sucrose,
50 mM Tris buffer, 1 mM EDTA, pH 7.4 (isolation media/homogenization buffer).
All subsequent steps are performed on ice to minimize tissue deterioration.
Obtain 2 ½ to 3 g of liver, and record the mass. Cut the liver into small pieces with
scissors to minimize core anoxia and necrosis. Use 35 mL of homogenization buffer to
quantitatively transfer the tissue into the chilled metal disruption container of the Omni-mixer.
Screw the container onto the Omni-mixer, lower the container into the ice, and disrupt the tissue
by setting the blade speed at 10,000 rpm for 30 s. Rest the sample for 1 min, then rehomogenize
at 10,000 rpm for another 30 s.
Filter the homogenate through cheesecloth to remove unhomogenized tissue. Measure
and record the total volume of the homogenate. (Tip: Filter the homogenate into a clean beaker
and then transfer to a graduated cylinder.) Save 0.1 mL of the homogenate in a microfuge tube.
This sample will be used to measure the amount of protein in the homogenate. (Tip: Remove
the 0.1 mL aliquot before transferring the homogenate into the graduated cylinder. ALWAYS
mix fractions to ensure a homogenous distribution before removing material.)
Part 2) Differential centrifugation - collection of three subcellular fractions:
Nuclear Pellet (NP), Mitochondrial Pellet (MP), and Post-mitochondrial Supernatant (PMS)
Safety Notice:
Any centrifuge can be dangerous - even deadly - if improperly used. Do not operate the
centrifuge without supervision. Always balance tubes.
Balance the centrifuge tube containing homogenate against another group's tube. Adjust
the weight by adding homogenization buffer to the lighter tube. Place samples in the SS-34 rotor
of the Sorvall centrifuge. DON'T FORGET TO BALANCE YOUR TUBES! Set the centrifuge
to run for 5 min at 600 x g. (Note: In the past, users would consult a nomogram (chart) to
determine appropriate rpm settings. Most centrifuges now automatically calculate the
appropriate run speed, based upon the identity (radius) of the installed rotor.)
The "eyeball-like" mass stuck to the bottom of the tube after this low-speed spin is called
the nuclear pellet (NP). Carefully decant the supernatant (don't let the "eyeball" slip out) into a
clean centrifuge tube and re-centrifuge at 10,000 x g for 20 min (BALANCE TUBES). The
brownish "bulls-eye" at the bottom of the tube following the second, high-speed centrifugation is
the mitochondrial pellet (MP). The supernatant is the post-mitochondrial supernatant (PMS).
Use a graduated cylinder to measure the volume, and then transfer into a plastic 50 mL storage
tube.
Both the nuclear and mitochondrial pellets (NP and MP) must be resuspended in
homogenization buffer. Add 5 mL buffer to each pellet and resuspend using a Pasteur pipette
and gentle vortexing. The success of the protein measurements depends on thorough mixing of
the pellets. Determine the total volume of these fractions and record in your notebook. Transfer
the NP and MP to separate plastic 15 mL storage tubes.
Part 3) Quantitation of protein in each fraction
The Bio-Rad Protein Assay will be used to measure the amount of protein in each
fraction. A new standard curve must be prepared. Refer to the "Lab Techniques" exercise.
The unknown samples (H, NP, MP, PMS) need to be diluted (1/20) so the amount of protein in
the dilution falls within the concentrations measured in the standard curve.
1) Obtain 9 glass test tubes. Label these H, NP, MP, PMS (fractionation samples) and 1 - 5
(BSA standards).
2) Make a table showing how much water and sample to add to each tube. Refer to the
"Laboratory Techniques" protocol for how to prepare the BSA standard curve.
3) Add 5 mL of Bio-Rad Dye Reagent to each tube. Add dye reagent to all tubes, both samples
and standards, at the same time. Vortex to mix.
4) Transfer (by pouring) the solutions into disposable plastic cuvettes.
5) Record the absorbance at 595 nm using the 0 µg BSA/µL standard as the reference.
Lab Report Write-Up Guide
Show sample calculations where appropriate.
In the results section of your lab report, determine:
1) the protein content (in mg) of each subcellular fraction (H, NP, MP, PMS). To determine the
protein content, first find the concentration of protein from the standard curve in mg/mL. Take
the dilution factor into account. If a 1/20 dilution was prepared, the protein concentration of the
dilute sample needs to be multiplied by 20 in order to determine the concentration of protein in
the original sample. Finally, multiply by the total volume of the fraction to calculate the total
protein present in the original sample.
Suggested figures and tables for point 1:
Graph : Bio-Rad Standard Curve
Table: Bio-Rad Standard Curve; Column Headings: Tube Number, BSA (mg/mL), A595 nm
Table: Fractionation Summary; Column Headings: Fraction, Dilution Factor, A595 nm, Protein
Concentration - Dilute (mg/mL), Protein Concentration - Original (mg/mL), Total Volume (mL),
Total Protein (mg)
Supply figures and tables, as appropriate, to summarize findings in points 2-4.
2) the percent protein per gram of tissue (% protein / g tissue) given the protein concentration of
the homogenate and the original tissue weight.
3) the total protein of NP+MP+PMS compared to the total protein of H. Theoretically, these
values should be the same. Determine the % error using the following approach:
The total protein of H is the expected value.
4) the percent protein of each fraction, per total protein recovered. Ex:
In your discussion section, consider:
1) possible reasons for your results. Why might there be a greater percentage of protein in one
particular fraction? It is important to explain the distribution of protein within the cell. Use
numbers from your results section to reinforce your statements.
2) discrepancies in the data. Compare the data to published information on subcellular
distribution of protein. Does the distribution found in lab agree? Are there similar trends? Also
evaluate data within the group. For example, the total protein found in the subcellular fractions
might add up to a number greater than the original tissue weight! It is fine if the results are
unusual, but unexpected results must be explained.
References
1) Lodish, et al., eds. (2000) Molecular Cell Biology, 4th ed. W.H. Freeman and Company. Figure 5-23.