Proteasome Preparation from Human Brain
Proteasome Preparation from Human BrainYou will need:-
Chicken gamma globulin (1mg/ml in H2 O). Chicken gamma globulin standards :-
Gamma Globulin Standards
| Final Quantity of Gamma Globulin | Gamma Globulin | H2 O |
|---|---|---|
| 5mg | 5ul | 45ul |
| 10mg | 10ul | 40ul |
| 20mg | 20ul | 30ul |
| 30mg | 30ul | 20ul |
| 40mg | 40ul | 10ul |
| 50mg | 50ul | Zero |
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).
Homogenising Buffer
| Component | Quantity |
|---|---|
| 500mM Tris | 4ml |
| 100mM ATP | 2ml |
| 50mM MgCl2 | 10ml |
| 50mM DTT | 2ml |
| Make up volume to 100ml with dd water |
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)
METHOD 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:-
Glycerol Gradient Preparation
| Final Concentration of Glycerol | Glycerol | Homogenising Buffer |
|---|---|---|
| 40% | 4ml | 6ml |
| 30% | 3ml | 7ml |
| 20% | 2ml | 8ml |
| 10% | 1ml | 9ml |
| 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:-
- a) Add 5ul sample (or 10ul if protein concentrations are low) to a 4ml tube. b) Add 100ul of working substrate solution. c) Incubate for 15 minutes at 37°C. d) Stop reaction with 2ml of stop solution. e) Read fluorescent emission @ 460nm with an excitation wavelength of 360nm on a fluorimeter. f) Plot emission against fraction number to show activity peaks.
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:-
- a) 50ul sample + 1/12 dilution of Bradford reagent. b) Stand for 2 minutes. c) Read OD @ 595nm. d) Repeat assay using standards i.e. Chicken gamma globulin at known concentrations.
N.B.: Fractions 1 to 11 - 10ul sample + 40ul water. Fraction 12 onwards 50ul undiluted sample.
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