Laboratory 6 Week 1 : Cell Fractionation
Introduction:
Eukaryotic cells are complex and contain many kinds of membrane organelles. For instance, they contain nuclei, mitochondria, vacuoles etc. Two methods exist to study the organelles in more detail. The first is by using a variety of techniques to visualize the nuclei while still inside the cells by microscopy (i.e. cell staining and immunofluorescence). The second method involves suspending the cells in solution, and breaking them open (lysing the cells). Then the various organelles are then separated from each other by centrifugation, which then allows them to be used for further study. It is this second method that we will use in this laboratory.
A procedure called cell fractionation is used to break open the cells and separate the various organelles. To perform cell fractionation, we first will suspend our cells in solution, and then we break open the cells, or lyse them. This will release the organelles inside into solution. Next, we can separate the organelles by centrifuging our solution. By using centrifugation, we can easily separate the various organelles, since the various organelles are of different mass, and density (for instance, nuclei are significantly heavier than mitochondria etc.). During centrifugation, different organelles will pellet at the bottom at specific speeds based on the mass and densities of the organelles. For instance, the heavier (larger) the organelles, the less velocity is needed to pellet the organelle. The lighter (smaller) the organelle, centrifugation must occur at a greater velocity to pellet the organelle.
Therefore, if we want to separate nuclei from mitochondria, we will centrifuge at low speed. At low speed, the nuclei will pellet at the bottom, while the mitochondria will stay suspended in the solution. The left over solution after centrifugation is called the supernatant, and will contain lighter organelles (i.e. mitochondria and dissolved proteins). If we then want to separate the mitochondria from the rest of the supernatant, we can centrifuge at the appropriate speed that would pellet the mitochondria, and then remove the resulting supernatant.
If we first centrifuged or cell suspension at the speed appropriate to pellet mitochondria, we would bring the mitochondria to the bottom of the tube. However, we would pellet everything that is heavier than the mitochondria (i.e. nuclei etc.). Therefore, in order to get nuclei separated from mitochondria, we must centrifuge at the lower speed first to obtain the nuclei, and centrifuge the supernatant at the higher speed to collect the mitochondria. This type of separation protocol is called differential centrifugation. In this procedure, it is possible to separate the organelles to purification because objects a similar size to our desired organelles will also pellet at the same speeds. However, these objects are significantly less concentrated in our suspension. Therefore, our pellets will are enriched for the organelle we are attempting to isolate.
Each pellet that is isolated can then be resuspended by adding solution, and can be called a fraction (for instance, the resuspended nuclear pellet is considered the nuclear fraction). Additionally, a sample of the original suspension is considered a fraction, and is called the crude fraction, as it contains all the organelles and soluble proteins. Lastly, a sample of the final supernatant is also considered a fraction and is called the soluble fraction, and contains everything that was not pelleted by centrifugation.
Using a Centrifuge
There are various types of centrifuges one can use. Various kinds of centrifuges are available: clinical centrifuges seldom run faster than 5,000 rpm but are able to readily sediment cells, nuclei or chloroplasts. High-speed centrifuges go as fast as 25,000 rpm and can sediment smaller organelles such as mitochondria. Ultracentrifuges can produce speeds of 60,000 - 75,000 rpm and can sediment membranous organelles such as microsomes and golgi components. The high speed and ultracentrifuges are always refrigerated and often operated in a vacuum to reduce the heat generated when a rotor is rapidly spinning in air.
To centrifuge your sample, one must first get the appropriate rotor and place it on the pin in the centrifuge. The rotor you choose to use must fit the tubes in which you are placing your cell suspensions. The pin is rotated by the centrifuge motor, thus spinning your rotor. The relative centrifugal force your cell suspension is subjected to is dependent on the speed you are spinning your rotor, as well as the radius of the rotor. You can think of the rotor radius as the distance your centrifuge tube is from the axis of rotation (the pin). In general, your centrifuge tubes are not placed in the rotor in a straight up and down fashion, but are placed at an angle, with the bottom of the centrifuge tube being further from the axis of rotation than the top. The force that that the organelles are being subjected to in order to pellet them is specified by the formula R.C.F. = 1.119x 10-5 (rpm)2r, where RCF is relative centrifugal force, rpm is the revolutions per minute of the rotor and r is the distance (in cm) of the particle from the axis of rotation. Given that the rotor radius is larger at the bottom of the rotor than the radius at the top of the rotor, the RCF will be different along the length of the tube. Can you determine whether the top or bottom of your centrifuge tube will be subjected to greater force? The radius used for our purposes is generally called r(average) and is usually the mean of the maximum and minimum possible radii. Note, many centrifuge rotors come with conversion charts between RCF (which is noted by the units in multiples of gravity (x g)) and rpm.
The laboratory
In the cell fractionation laboratory, we will use differential centrifugation to create fractions enriched for specific organelles. We will first suspend whole plant cells in a mild salt solution (mannitol grinding medium) and lyse them using a mortar and pestle. A sample of the resulting solution will be the crude fraction. By doing differential centrifugation, we will obtain nuclear, mitochondrial and soluble fractions as well. The soluble fraction will contain objects such as soluble proteins, ribosomes and nucleic acids.
Materials
1. Cauliflower or Spinach
2. Razor blades (7)
3. Balances
4. Weighing Dishes (located next to balances in both 284 and 286)
5. Mortar and Pestles (In Cold Room – Chilled)
6. Grinding Sand (Next To Balance in 286)
7. 50 mL Graduated Cylinders (24)
8. Paring knives (2)
9. Ice buckets (6)
10. Oak Ridge Tubes (12) – Chilled in Cold Room
11. Cheesecloth
12. Scissors (3 pairs) – near cheesecloth
13. 250 mL beakers (12)
14. Microfuge Tubes
15. Small glass stirring rods
16. Microscope slides
17. Microscopes
18. Coverslips
19. Methyl Green Pyronin
20. Spectrophotometers
21. 5 mL Microcuvettes
22. Ziplock Bags
23. 13 X 100 test tubes (40)
24. Distilled water (4 x 100 mL bottles)
25. Sharpies (8)
26. Bin for dirty dishes
27. Bin for dirty cuvettes
28. Protein solution (0.1 mg/ml)
Make 2 bottles/100 mL
For 200 mls, take a 400 mL beaker
Fill Beaker with 100 mL deionized water
Weigh out 1 g of BSA and mix into the water
Bring volume up to 200 mL and aliquot into 100 mL bottles
29. Mannitol Grinding Medium
Use a 1 L flask and add 850 mL Deionized water
Add 54.66g D-Mannitol
Add 0.82g KH 2 PO 4
Add 2.42g K 2 HPO 4
pH to 7.2 (This step is extremely important-BE ACCURATE!)
Bring volume up to 1L with deionized water and aliquot in 500 mL bottles (2)
Store Media in the Cold Room
30. Mannitol Assay Medium
Use a 600 ml beaker and add 400 mL Deionized water
Add 27.33 g D-Mannitol
Add 0.41 g KH 2 PO 4
Add 1.21 g K 2 HPO 4
Add 0.38 g KCl
Add 0.51 g MgCl 2 X 6 H 2 O
pH to 7.2 (This step is extremely important – BE ACCURATE!)
Bring volume up to 500 mL with deionized water and aliquot into 2 250 mL Bottles
Store media in the Cold Room
31. 0.4 M Perchloric Acid
Take 34.5 mL concentrated Perchloric Acid
Bring volume up to 1 L with deionized water
32. Protein Dye solution
Obtain a 1 L beaker
Place 50 mL of 95% ethanol in the beaker
Add 600 mg of Brilliant Blue G
Add 750 mL Perchloric Acid and Mix
Bring volume up to 1L with deionized water
Experimental Protocol
Part A: Fractionation of Cauliflower or Spinach Cells (1 head of cauliflower or bag or spinach per lab section)
1. Using a single-edge razor blade, remove a total of 20 g of the outer 2-3 mm of the cauliflower surface. [Alternate: Whole class works together to remove a total of about 220 g of the outer 2-3 mm of the cauliflower surface. See pictures under the “Results” hyperlink.]
2. Place the cauliflower tissue in a chilled mortar with 40 mL of ice-cold mannitol grinding medium and 5 g of cold purified sand. Grind the tissue with a chilled pestle for 4 minutes (on ice!). [Alternate: Whole class puts the entire 220 g of cauliflower tissue into the kitchen blender. 300 mL of the ice-cold mannitol grinding medium is poured on top. The blender is “pulsed” several times to break up the largest chunks of cauliflower tissue. The cauliflower is then homogenized at “liquefy” speed for 1 minute. See pictures under the “Results” hyperlink.]
3. Filter the suspension through four layers of cheesecloth into a beaker (also wring out the juice); measure and note the volume, then save a small measured amount (~ 2-3 mL) in two microfuge tubes for assays and microscopy. Label the “crude” fraction. Transfer the rest to a chilled 40 mL centrifuge tube. Keep the centrifuge tubes on ice until all groups are ready to centrifuge.
4. Centrifuge the filtrate at 600 x g (4200 rpm, JA20 rotor; 4000 rpm, SS34 rotor) for 10 min at 4° C. Make sure that the centrifuge tubes are balanced; the tubes opposite each other should have the same total volume. Place a 50 mL graduated cylinder on ice. The pellet from this centrifugation is the nuclear pellet.
5. Decant the supernatant from the centrifugation into the chilled graduated cylinder (save the nuclear pellet on ice). Measure the supernatant volume and pour the supernatant into a clean 40 mL chilled centrifuge tube. Spin at 10,000 x g (17,000 rpm, JA20 rotor; 16,000 rpm SS34 rotor) for 20 minutes at 0° - 4° C. Again, be sure that the centrifuge tubes are balanced. Meanwhile, resuspend the nuclear pellet in 5 mL mannitol assay medium. Put another 50-mL graduated cylinder on ice.
6. After the 20 minute centrifugation, transfer the supernatant to the graduated cylinder; note the volume and transfer to a clean tube (Label “soluble” fraction). Resuspend the mitochondrial pellet in 8.0 mL of ice- cold Mannitol Assay Medium. (Use a glass rod or a Pasteur pipette; make sure all the clumps are completely dispersed). Label “Mitochondria”. Store all tubes on ice until the end of the period.
7. You now have 4 samples: crude, nuclear, soluble, mitochondrial.
8. Prepare wet mounts of each and examine by microscopy. Capture a representative image at 400X of each preparation for your notebook. Record observations of the kinds of particles present and their relative frequencies.
9. Prepare slides of all four preparations with methyl green pyronin:
· Place a small drop of each fraction on a clean slide
· Add a drop of stain
· Cover with a coverslip
· Observe at 400X with bright field: nuclei should stain green, cytoplasm red or pink, and mitochondria can be seen as small dots.
· Capture an image of all four preparations for your Results Section
10. Measure the protein concentration in each aliquot as described below and record the concentrations in your notebook. You will need the protein concentrations later to report the specific activity of your preparations.
11. After observing each fraction microscopically and measuring the protein concentration of each fraction, you will need to freeze the fractions for use next week. Put about 1 mL of the crude, nuclear, soluble, and mitochondrial fractions into labeled 1.5 mL microfuge tubes. Put ALL of the remaining mitochondrial suspension (about 7.0 mL) into a labeled sterile 10 mL plastic tube with a screw cap. This larger amount of the mitochondrial fraction will be needed for activity assays next week. Write the names of all group members as well as the day and time of your lab on the outside of a small Ziploc bag. Put all your labeled samples in the small Ziploc bag. Place all of the small Ziploc bags into a large Ziploc bag labeled with the day and time of the lab. Store the large Ziploc bag in the cell biology freezer.
Part B: Determination of Protein Concentration of Your Fractions
Standard Curve for Protein Concentration Determination
Proteins are necessary for cells to function properly. Without them, cells would not be able to have the proper structure, and would not be able to carry out important processes for life. Additionally, the different organelles have different amounts and different types of proteins. Therefore, our different fractions will contain different amounts of protein.
In order to determine how much protein is in each fraction, we must create a standard curve by using protein solutions of known concentrations and determining their absorbance using the spectrophotometer. This standard curve will then be used to determine the protein concentrations in your fractions. A protein that is frequently used for standard curves is Bovine Serum Albumin (BSA). BSA readily dissolves in water to form a colorless solution. Dissolved BSA reacts with specific dye known as Coomassie Blue G-250. Your resulting solutions will turn various shades of blue depending on the concentration of protein that is in your sample. If your sample has a high protein concentration, your solution will turn deep blue. If your solution has a low protein concentration, it will turn a brownish blue. If your solution has no protein in it, then it will be brown.
In this lab you will: A) mix known concentrations of BSA dissolved in buffer with Coomassie Blue G-250, B) measure the absorbance of the resulting blue colored solution and C) plot the measured absorbance of the protein-dye conjugate versus the known concentration of BSA in that sample. The resulting graph will be the protein standard curve that you will then use to determine the amount of protein in your samples of unknown protein concentration.