Proteins, composed of chains of amino acids joined together by peptide bonds, are one of the building blocks of life. They have many different functions and come in every imaginable size, conformation and structure. Researchers have been routinely isolating proteins to analyze their structure and function. Isolation of protein is relatively easy but the specific steps depend on the tissue or organism of interest. Beef liver is a cheap and easy tissue to begin with to practice protein isolation.
Preparation of Tissue
Step 1
Remove the beef liver from the package and place on a clean cutting board or work surface.
Step 2
Cut a 1 inch by 1 inch portion of the liver using a clean knife or razor blade.
Step 3
Inspect the section of tissue and remove pieces of fat or other connective tissue.
Step 4
Mince the tissue into small pieces and place into the container of a prechilled blender.
Step 5
Measure 75 ml of prechilled protein extraction buffer with a clean measuring cup or graduated cylinder and pour into the blender.
Extraction of Protein
Step 1
Put the chilled blender on the base, cover, and blend for four 30-second bursts.
Step 2
Pour the blended tissue into centrifuge tubes, making sure to pour the same amount into each tube to ensure a balanced centrifuge.
Step 3
Place the centrifuge tubes into the rotor of the centrifuge, arranging them across from each other to keep the centrifuge balanced.
Centrifugation
Step 1
Turn on the centrifuge to 15,000 rpm for 20 minutes.
Step 2
Wait for the centrifuge to stop moving and carefully remove the tubes, making sure not to bump them on the way out.
Step 3
Place the centrifuge tubes in the refrigerator until ready for filtration.
Filtration
Step 1
Place several layers of the cheesecloth into the funnel.
Step 2
Slowly pour the supernatant (the clear fluid portion) through the cheesecloth into a chilled clean beaker or glass.
Step 3
Discard the pellet (the clump remaining in the bottom of the tube), which contains membranes, nuclei and cellular debris.
Step 4
Place the beaker containing the filtered homogenates into the container of ice.
Precipitation
Step 1
Stir the extract very slowly with a clean stirring instrument while it is submerged in the container of ice.
Step 2
Add the powdered ammonium sulfate very slowly while continuously stirring.
Step 3
Continue stirring for several minutes while the mixture is in the container of ice until the ammonium sulfate has completely dissolved.
Step 4
Pour the mixture into two clean centrifuge tubes, making sure to pour the same amount into each tube.
Protein Centrifugation
Step 1
Place the centrifuge tubes into the rotor of the centrifuge as before.
Step 2
Centrifuge the tubes at 15,000 rpm for 20 minutes.
Step 3
Pour off and discard the supernatant gently, leaving behind the pellet. The pellet stuck to the bottom of the tube contains the crude protein extract from the original liver tissue.
Tips and Warnings
- The only specialized equipment needed for this procedure is a centrifuge and centrifuge tubes. These are rather expensive but can be found in almost any basic biological laboratory.
- The protein extraction buffer and ammonium sulfate are dangerous and should not be touched with bare hands. Make sure to wear gloves and protective goggles throughout the entire procedure. Centrifuges can be very dangerous if used improperly. Do not put your hand into the centrifuge until the rotor has completely stopped turning. Also, make sure to properly balance the centrifuge before using it.
Things You'll Need
- Fresh beef liver
- Cutting board
- Sharp knife or scalper
- Blender
- Refrigerator
- Cheesecloth
- Funnel
- Clean beakers or glass cups
- Centrifuge
- Centrifuge tubes
- Protein extraction buffer
- Small measuring cup or graduated cylinder
- Ammonium sulfate powder
- Large container of ice
- Clean stirring instrument
- Rubber gloves
- Goggles
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