Chicken Liver - Homogenization and Centrifugation
I had no idea there were so many things you could do with chicken liver in the lab.
Purpose: Obtain, Homogenize and Centrifuge chicken liver for protein extraction.
Materials:
Gloves
Scissors
50mL Beaker
Homogenization Buffer (1/10/13 JNKV)
Centrifuge tubes
High Speed Centrifuge (Sorvell RCB5 & SS34 Rotor)
Ice Bucket
Ice
(3) 1.5mL Minifuge tubes
Minifuge tube rack
Homogenizer
2X SDS-PAGE
2.85g Chicken Liver
p200 (Set 7)
KimWipes (A Lot)
Procedure:
- Filled large ice bucket with ice, 2 centrifuge tubes (to cool), 3 labeled minifuge tubes in tube rack and scissors.
- Obtained approximately 2.85g Liver & chopped into fine pieces (Actually, I let my lab partner do this. She's a champ!)
- Poured chopped liver into 15mL homogenization buffer stirring SLOWLY in beaker on ice on stir plate.
- Transferred buffer and liver to homogenizer.
- Used the loose pestle, then the tight pestle to (gross myself out completely and) homogenize to smooth consistency, then transferred to centrifuge tube.
- Weighed and balanced sample in centrifuge tube (29.6g).
- Extracted 50microL sample (before weighed and balanced) for SDS PAGE samples in minifuge tube
- Centrifuged in pre-chilled SS-34 Rotor 600xG at 4 degrees C for 5 minutes (2200RPM) on Sorvell RCB5. Rotor Calculations: RCF=11.2 x r(RPM/1000)^2. Where max radius for SS34 rotor is 10.7cm.
- Put 2nd 40mL centrifuge tube on ice and ready for transfer of supernatant.
- Put centrifuged sample immediately into ice bucket.
- Used 10mL pipette to extract supernatant from tube without disturbing the loose pellet.
- Transferred supernatant into the pre-chilled 40mL centrifuge tube. There wasn't a noticeable white layer of fat.
- Weighed and prepared a counter balance for the new centrifuge tube (29.1g)
- Added a 50micoL sample to the "Liver Extract 2" tube and added to microliters of SDS PAGE. Then added another 50 microliters of SDS PAGE to "Liver Extract 3" tube so it's ready to go when the next spin is over.
- Centrifuged 2nd tube for 10 minutes at 6000xG (RPM 7100 for SS34 rotor).
- Added clean 50mL conical tube to ice.
- After centrifugation, took 50 microliter sample of supernatant and added it to "Liver Extract 3" microfuge tube already containing 50 microliters of SDS-PAGE.
- Measured balance of supernatant, (8.9mL) for calculation to get amount of needed ammonium sulfate.
Observations and Results:
Final supernatant collected was 8.9mL. Using ammonium sulfate calculator (http://www.encorbio.com/protocols/AM-SO4.htm), we determined 2.06g of ammonium sulfate required for 40% range and final volume of 9.99mls.
Conclusion:
Centrifuge use is SERIOUS business. Here are some of the notes from our orientation with the Sorvell:
Never use the centrifuge alone.
Never use the centrifuge with a lab partner without notifying an instructor you're using the centrifuge.
This is a fixed angle rotor which means the pellet will form as a slant.
Never use a rotor made for another brand's centrifuge.
A rotor's max speed is degraded over time by inspection technicians based on it's age/use.
Always use the log book so inspections are as accurate as possible.
Always leave the brake on unless otherwise noted in your protocol.
Don't line the pins on top of each other.
Check tube compatibility for material, speed and rotor.
Check tubes for stress fractures. Don't use if there's a fracture.
Make sure there's a cup cushion in each rotor hold.
Always make sure your tubes are balanced by weight not by volume.
Tighten the bottom screw first (attaches rotor to centrifuge), then tighten lid to rotor. Spin the rotor against the mechanism to make sure very tight.
Centrifuge tightening is counterclockwise.