Appendix 2:  Preparation and Storage of Dogfish Erythrocyte Cytoskeletons.

A.  APPENDIX 2 PROCEDURE

(for erythrocytes obtained from 80 ml blood, as in Appendix 1)

NOTE : For solutions needed, see section B below; WS = Working Solution; WS#5 = Brij Lysis medium; WS#6 = 25% Glycerol Gradient Solution; WS#7 = 40% Glycerol Gradient Solution; WS#8 = 50% Glycerol Gradient Solution

NOTE: Pre-warm all solutions to ~20-23°C before use.

Centrifuge - We use the International Equipment Corporation Model CS, equipped with #277 4-place rotor that holds 200ml conical-bottom plastic bottles.

Starting with 600-ml suspension of washed erythrocytes in Ringer's:

  1. Prepare eight glycerol step gradient bottles as follows - first add 10 ml 50% glycerol gradient solution (WS#8) to the bottom of the centrifuge bottle; then gently layer 70 ml of the 40% glycerol solution (WS#7) onto it, and finally 40 ml of the 25% glycerol solution (WS#6).
  2. Centrifuge erythrocyte suspension in Ringer's (75 ml/bottle) Siphon off any remaining white cells and resuspend cells up to 600 ml with Brij LM (WS#5).
  3. Layer 75 ml cytoskeleton suspension onto each glycerol gradient
  4. Centrifuge for 10 minutes at ~2400 RPM (~1,300g. Cytoskeletons will sediment, producing a loose white pellet at the bottom.
  5. Siphon off red layer (down to approx. where bottle begins to taper, leaving ~ 40 ml). Do not yet go to the level of the pellet, or hemoglobin will contaminate the preparation.
  6. Rinse sides of tube by dribbling K-PEM or Na-PEM completely around its internal circumference, allowing remaining hemoglobin to wash into a layer above the glycerol. Aspirate that layer plus the remaining glycerol gradient solution above the pellet, and discard.
  7. Resuspend pellets in 50% glycerol-KPEM (=WS#8) or 50% glycerol-NaPEM if used in the gradient. Pool and bring total volume to 40 ml.
  8. If the cytosketons are to be used directly, the suspension can be diluted with Na-PEM or K-PEM to reduce the glycerol concentration, and the cytoskeletons sedimented by gentle centrifugation.
  9. If long term storage is desired, with preservation of marginal bands, add taxol to 10µM final concentration (we add 40 µl stock 10 mM taxol in DMSO to 40 ml cytoskeleton suspension), incubate ~20 min at room temp. (~22°C), and store in the freezer at -20°C.

(B) APPENDIX 2 RECIPES - WORKING SOLUTIONS = WS; STOCK SOLUTIONS= SS

SS#6 , = 1M MgCl2 (100ml)

INGREDIENT(S) AMOUNT/VOL FINAL CONC.
MgCl2.6H2O 20.33g 1 M
H2O to 100ml -

SS#7, = 2X K-PEM (1 liter)

INGREDIENT(S) AMOUNT/VOL FINAL CONC.
PIPES 60.48 g 0.2 M
EGTA 3.80 g 0.01 M
MgCl2 2 ml SS#6 0.002 M
H2O to 1000ml -

NOTES

  1. Mix ingredients in 1 liter beaker with ~800 ml deionized water;
  2. Add KOH (as pellets initially; later as a solution in water) until pH 6.8 is reached; solution will be turbid initially, and will clear when sufficient KOH is added.
  3. Bring final volume up to 1 liter with deionized H2O.
  4. Storage conditions - FREEZE

SS#8 2X Na-PEM (1 liter)

INGREDIENTS - Same as for 2X K-PEM (SS#7) above, except use NaOH to adjust to pH 6.8.

NOTES
(1-4) As for SS#7, above (except using NaOH)
(5) Na-PEM can replace K-PEM in all solutions when cytoskeletons or fractions prepared from them are to be used for SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE); this eliminates precipitation of SDS by K+.

WS#5, BRIJ Lysis Medium - TOTAL VOLUME TO BE PREPARED = 600ml

ADDITIONAL STOCK SOLUTIONS NEEDED

STOCK SOL'N # INGREDIENT(S) AMOUNT/VOL
SS#9 TAME 25g/660 ml
SS#10 BRIJ 30g/500 ml
SS#11 Pefabloc 0.1g/100 ml
SS#12 Leupeptin 5mg/1 ml
SS#13 Pepstatin 5mg/7 ml (in methanol)
SS#14 Aprotinin 10mg/10 ml

NOTES
(1) All stock solutions except SS#11 should be stored frozen. Pefabloc should be refrigerated.
(2) Pefabloc (Boehringer Mannheim Corporation, Indianapolis, IN) is used instead of PMSF (used in earlier work) because it is much more stable in solution, and thus solutions containing it can be prepared in advance. It is, however, much more expensive than PMSF. The procedure compensates for this increased cost to some extent by greatly reducing the amount of PIPES buffer required (as compared with earlier published procedures; Cohen and Ginsburg, 1986)
(3)Leupeptin, Pepstatin A, Aprotinin - we use Sigma # L2884, #P4265, and #A1153.

FOR WS#5, BRIJ Lysis Medium

INGREDIENT(S) AMOUNT/VOL FINAL CONC.
2X K-PEM SS#7 300 ml 1X K-PEM
TAME stock SS#9 60 ml 10 mM
BRIJ stock SS#10 60 ml 0.6%
Pefablock stock SS#11 14.5 ml 24 µg/ml
Leupeptin stock SS#12 60 µl 0.5 µg/ml
Pepstatin stock SS#13 600 µl 0.1 µg/ml
Aprotinin SS#14 60 µl 0.1 µg/ml

NOTES
(1) Storage conditions - mix first three ingredients and FREEZE.
(2) Protease inhibitors (SS# 10-13) should be added shortly before use; bring final volume up to 600 ml.

WS#6, 25% Glycerol Gradient Solution; TOTAL VOLUME TO BE PREPARED = 350 ml

INGREDIENT(S) AMOUNT/VOL
2x K-PEM SS#7 (or SS#8) 175 ml
Glycerol 87.5 ml
TAME SS#9 35 ml
BRIJ stock SS#10 35 ml
Pefabloc stock SS#11 8.5 ml
Leupeptin SS#12 17.5 µl
Pepstatin SS#13 175 µl
Aprotinin SS#14 17.5 µl

NOTES
(1) Add protease inhibitors (SS# 10-13) before use and bring up to 350 ml with deionized H2O
(2) Add 40 ml/200 ml plastic centrifuge bottle using a funnel.
(3) Storage conditions - FREEZE

WS#7, 40% Glycerol Gradient Solution; TOTAL VOLUME TO BE PREPARED = 600 ml

INGREDIENT(S) AMOUNT/VOL
2x K-PEM SS#7 (or SS#8) 300 ml
Glycerol 240 ml
H20 60 ml

NOTES
(1) Storage conditions - FREEZE
(2) Add 70 ml/200 ml plastic centrifuge bottle using a funnel.

WS#8, 50% Glycerol Gradient Solution; TOTAL VOLUME TO BE PREPARED =100 ml

INGREDIENT(S) AMOUNT/VOL
2x K-PEM SS#7 (or SS#8) 50 ml
Glycerol 50 ml

NOTES
(1) Storage conditions - FREEZE
(2) Add 10 ml/200 ml plastic centrifuge bottle using a funnel.