Chicken gamma globulin (1mg/ml in H2O).
Chicken gamma globulin standards:-
|Final Quantity of Gamma Globulin||Gamma Globulin||H2O|
Make up the Homogenising Buffer (3ml/g tissue) in the following way:-
Tris 500mM = 30.3g/500ml. Adjust to pH7.5 with HCl.
ATP 100mM = 0.551g/10ml. Adjust to pH7.5 with NaOH (can be stored at -20°C).
MgCl2 50mM = 1.02g/100ml.
DTT 50mM = 0.771g/100ml (prepare fresh).
|Make up volume to 100ml with dd water|
Working chymotrypsin substrate solution
70ul 100x stock in 7mls 100mM Tris (pH7.5)
80mM Sodium acetate pH 4.3 (2.04g NaAc, in 500ml dd water, pH with glacial acetic acid)
2) Centrifuge homogenised sample @ 10,000rpm for 20 minutes in Europa 8 x 50 (or equivalent).
3) Decant supernatant (5ml/tube) onto a glycerol gradient. The gradient is prepared in the following manner:-
|Final Concentration of Glycerol||Glycerol||Homogenising Buffer|
|Gently load 7ml of each % into MSE tube for 6x38 swing out rotor (highest % first)|
4) On top of gradient, load 5ml supernatant.
5) Balance tubes and spin at 23,000 rpm @ 4°C in a MSE65 prepspin (or equivalent) for 22 hours.
6) Displace 1ml fractions from gradient using Maxidens (this is very dense and when pushed into the tube, displaces the gradient gently), giving approximately 35 x 1ml fractions.
7) Assay fractions for protein and chymotrypsin (or other peptidase) activity:-
8) Bradford assay for protein:-
N.B.: Fractions 1 to 11 - 10ul sample + 40ul water.
Fraction 12 onwards 50ul undiluted sample.
Department of Biochemistry,
University of Nottingham,
The Medical School ,
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