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.
