Dominik Haudenschild August 10, 1993 at CBRC

• Clean 15 7-8 week old sterna:
- Cut light tan cartilage away from darker, bloody bone, discard bone
- Remove any areas with blood, discard bloody parts
- With toothed forceps, completely peel away perichondrium on all faces sterna
- Freeze on dry ice, and weigh (assume 1ml ~ 1g)

• Grind frozen sterna in coffee grinder with about equal volume dry ice

• Extract in 15 volumes 4M Guanidine (buffer #2) overnight 4°C with stir bar

• Spin 10000rpm in GSA to clear solution, ~10-15 minutes (pellet contains mostly cross-linked collagen, most proteoglycans are gone)

• For associative conditions dialyze against 10 volumes buffer 2 without Guanidine (for 0.4M final guanidine concentration). After dialysis clear solution as in previous step

• Weigh extract to 1/10 gram precision

• For associative gradient add 1.19g Cesium Chloride per 1.00 gram extract, carefully swirl solution to allow Cesium Chloride to dissolve
Initial density of 1.7g/ml
-check by weighing 100µl in pipette tip (=0.17g/100µl)

• For dissociative gradient add 0.59g Cesium Chloride per 1.00 gram extract, carefully swirl solution to allow Cesium Chloride to dissolve
-Initial density of 1.5g/ml
-check by weighing 100µl in pipette tip (=0.15g/100µl)

• Put into appropriate ultracentrifuge for 48 hours at 4°C
36,000rpm in Ti50 rotor (~12ml/tube)
30,750rpm in 50.2Ti rotor
32,300rpm in Vti50 rotor

• Carefully remove protein pellicle if associative gradient

• Carefully pipette six equal fractions from the top of tube:
(fraction 6 is on top, fraction 1 is on bottom )

• Weigh 100µl of each fraction to confirm linear gradient (optional, but fun)

• To run another dissociative gradient, follow these directions:

• Make sample density 1.50g/ml (=0.696g CsCl per ml) by dilution with buffer 2 or addition of more CsCl as follows:

• Weigh 100µl of sample to get its density

• Get corresponding mg/ml CsCl from solution density on table

• Calculate total g of CsCl in sample by multiplying:
(mg/ml) x (total volume)

• Calculate additional amount of CsCl necessary to reach 0.696g CsCl per ml

• Spin as before

• To run another associative gradient, follow these directions:

• Make sample density 1.70g/ml (=0.951g CsCl per ml) by dilution with buffer 1 or addition of more CsCl as follows:

• Weigh 100µl of sample to get its density

• Get corresponding mg/ml CsCl from solution density on table

• Calculate total g of CsCl in sample by multiplying:
(mg/ml) x (total volume)

• Calculate additional amount of CsCl necessary to reach 0.951g CsCl per ml

• Spin as before

Solution #1 Guanidine HCl 0.4M
Sodium Acetate 50mM
Disodium EDTA 10mM
6-AminoCaproic Acid 100mM
Benzamidine HCl 5mM
pH to 5.8

Solution #2 Guanidine HCl 4.0M
Sodium Acetate 50mM
Disodium EDTA 10mM
6-AminoCaproic Acid 100mM
Benzamidine HCl 5mM
pH to 5.8

Solution #3 Guanidine HCl 8M
Sodium Acetate 100mM
Disodium EDTA 20mM
6-AminoCaproic Acid 200mM
Benzamidine HCl 10mM
pH to 5.8

Solution #4 NaCl 0.5M
Sodium Acetate 50mM
Disodium EDTA 20mM
6-AminoCaproic Acid 200mM
Benzamidine HCl 10mM
pH to 5.8