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Proteasome Preparation from Human Brain

Proteasome Preparation from Human Brain

You will need:-

Chicken gamma globulin (1mg/ml in H2O).
Chicken gamma globulin standards:-

Bradford reagent:-
Dissolve 100mg coomassie G250 dissolved in 50ml 100% ethanol, add 100ml concentrated perchloric acid and make up to 200ml with water.

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).

Chymotrypsin substrate stock (100x)
10 mM N-Suc-Leu-Leu-Val-Tyr-7 amidomethyl coumarin (5mg/650ul DMSO).

Working chymotrypsin substrate solution
70ul 100x stock in 7mls 100mM Tris (pH7.5)

Stop solution
80mM Sodium acetate pH 4.3 (2.04g NaAc, in 500ml dd water, pH with glacial acetic acid)


1) Take the brain sample and homogenise in buffer.

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:-

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:-

N.B.: The chymotrypsin activity of the proteasome recognizes the bond between Tyr-7 amido, therefore cleaving the substrate amidomethyl coumarin, releasing a fluorescent product.

8) Bradford assay for protein:-

N.B.: Fractions 1 to 11 - 10ul sample + 40ul water.
Fraction 12 onwards 50ul undiluted sample.

Keywords: proteasome, brain

Submitted at 13:52 on 22/2/96 by:

Jill Fergusson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Q.M.C. ,
Clifton Boulevard,
NG7 2UH,

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