Lab Manual
biological reagents
Cell Free Systems
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misc methods

> Procedure for Preparing Microsomal Membranes from Dog Pancreas


Procedure for Preparing Microsomal Membranes from Dog Pancreas

Day before:

  • prepare all reagents
  • bake required glassware (3 beakers, several 9" Pasteur pipettes, 2-3 graduated cylinders, homogenizing cups)
  • reserve centrifuges
  • place Ti50.2 rotor into cold room
  • Weigh out PMSF and seal it in a tube with the final volume marked on it so that you can just add the ethanol on Dog day.

Dog day:

  • pre-cool centrifuge to 4EC
  • carry out all steps in cold room on ice
  • work as fast as possible (time to final spin should be <3 hrs)
  • take out DTT; reconstitute PMSF
  • Excise the pancreas and store in 100 ml of buffer A on ice (in a preweighed beaker to estimate weight).
  • Remove to a fresh beaker of buffer A (.100 mL) and add 1 ml of PMSF for 100 ml buffer. Swirl to mix.
  • Place some of the tissue on a glass plate and trim away the fat and connective tissue using razor blades.
  • Mince the tissue more finely (but not too much) with razor blades.
  • Place 4 mL per gram of tissue (maximum of 200 mL) of buffer A + 1 ml per 100 of PMSF and 1/1000 DTT into a fresh beaker and add the tissue to this as you finish with it (for weight estimate - always subtract 10-15 g for waste).
  • Homogenize about 50 ml at a time with 8 strokes (up + down is 2 strokes) of a motorized homogenizer (setting #6).
  • Place the homogenate into 40 ml centrifuge tubes. Balance the tubes.
  • Spin at 4EC, 600 x g for 10 min in JA-20 rotor (.2200 rpm).
  • Pellet is intact cells, some mitochondria and cellular debris. Discard. Collect the supernatant into fresh centrifuge tubes by carefully decanting so as not to disturb the pellet. Balance by weighing.
  • Respin at 10,000 g for 15 min (4EC) again using JA-20 rotor (.9,500 rpm).
  • Pellet is more mitochondria. Discard. Decant (carefully) supernatant into Ti50.2 rotor tubes, spreading it equally into an even number of tubes.
  • Place 60-80 ml of buffer B plus PMSF and DTT into a graduated cylinder and mix. Layer 8.0 ml of this sucrose cushion (buffer B) underneath the supernate in each tube (success to this is do it slowly - use the 9" pipettes, baked). Balance the tubes.
  • Spin in Ti50.2 rotor at 45,000 rpm (150,000 g) 4EC, for 4 hours.
  • Just prior to end of spin, prepare 100 ml buffer C plus 100 :l DTT. Mix. Turn on spec.
  • Aspirate the supernatant. Keep the pellet. Using the end of the small homogenizer probe, scrape the pellets from each tube and pour quickly into the small homogenizer tube containing 2 ml of Dog C buffer (+ DTT). Homogenize manually with 2 strokes. Homogenize each pellet separately then transfer to a 50 cc polypropylene tube. Pool all pellets into this tube. Rinse each centrifuge tube again with 1 ml of C, rinse the homogenizer tube last with 5 x 2 ml C. Dilute preparation to 40 ml then assay for further dilution.
  • Assay to determine how much to dilute. Dilute the membranes to 50 u A280/ml. That is, 1 :l (use the 1-10 :l Eppendorf pipettor to measure 1 :l of membranes, use sterile tips) of membrane suspension in 1 ml 0.1% SDS should give an OD(280 uv) of 0.05. Dilute with buffer C accordingly.

e.g. if OD = 0.210 then 0.210 ÷ 0.05 = 4.2
Therefore, dilute membranes 1:4 in buffer C.
Make sure you use the quartz cuvettes and blank the machine with 0.1% SDS.