LysosomeIsolationinIsotonicSucrose

LEVEL I

MATERIALS

• Rat liver
  

• Physiological saline (0.85% w/v NaCl)
  

• 0.25 M sucrose in 10 mM Tris-HCL, pH 7.4
  

• Brendler teflon homogenizer
  

• Refrigerated preparative centrifuge
  

• 0.08 M CaCl in 0.25 M sucrose plus 10 mM Tris-HCl
  

• 150 mM KCl in 10 mM Tris-HCl Buffer, pH 7.4
  

• Phase contrast microscope, slides, coverslips

PROCEDURE 4

1. Decapitate and exsanguinate a rat that has been starved for at least 24 hours prior to the lab. 5

   Fill a syringe with saline and gently perfuse the liver by forcing the saline through the hepatic portal vein, and through the liver.

   
   

2. Remove the liver, place it in a preweighed beaker and weigh the beaker and liver. Calculate the weight of the liver.

   
   

3. Prepare a 10% (w/v) homogenate or brei. For each gram of liver, add 9.0 ml of 0.25 M sucrose in 10 mM Tris-HCL, pH 7.4. to the beaker.

   
   

4. Gently chop the liver in the sucrose and transfer the chopped liver to a teflon homogenizer. 6

   Gently homogenize the liver while keeping it chilled.

   
   

5. Centrifuge the brei at 12,000 xg for 10 minutes at 4 ° C. Decant the supernatant into a chilled beaker and discard the pellet.

   
   

6. Add 0.08 M CaCl to the supernatant to yield a final concentration of 8 mM (use 1 ml of CaCl per 9 ml of supernatant). Stir gently and recentrifuge at 25,000 xg for 15 minutes at 4 ° C

   
   

7. Carefully remove and discard the supernatant. 7

   
   

8. Resuspend the pellet (containing the lysosomes) in 30 ml of 150 mM KCl in 10 mM Tris-HCl Buffer, pH 7.4.

   
   

9. Re-sediment the lysosomes by a final centrifugation at 25,000 xg for 15 minutes at 4 ° C.

   
   

10. Remove a small portion of the pellet for Exercise 7.2. Resuspend the remainder of the pellet in 30 ml of 150 mM KCl/10 mM Tris-HCl Buffer. This suspension is the lysosome fraction for Exercise 7.3.

    
    

11. Prepare a wet mount of the resuspended lysosome pellet and observe with a phase contrast microscope at 100X. Draw any structures observed.