Activation of apoptotic activity in cytoplasmic extracts and

Monday, November 24, 2003

Description
Activation of apoptotic activity in cytoplasmic extracts and measurement of the resulting caspase enzymatic acticity

Procedure
Activation reaction

Cytoplasmic extracts were activated according to following protocol (as an example, cytochrome c/dATP are used as apoptotic stimuli). The components were mixed in the given order and then incubated at 37C usually for 45 min.

x l Dilution Buffer (DB) containing ATP regeneration system +
1.5 l cytochrome c from bovine heart (50 M) +
1.5 l dATP (10 mM)+
y l cytoplasmic extract (37.5 g protein)

x was so adjusted that the total volume was 15 l and thus the final protein concentration at 2.5 g/l.

When active recombinant caspases (such as caspase-8) are used for activation of the extracts, 100 ng of recombinant caspase is added instead of cytoc/dATP.

In case that the effect of inhibitors was studied, such as Ac-DEVD-fmk, or zVAD-fmk, the inhibitor at concentrations ranging from 100 nM up to 100 M was preincubated together with the extract in DB for 5 min, then cyto c/dATP was added.

Caspase enzymatic assay

After incubation of the extracts in the presence or absence of activating stimuli (cytochrome c/dATP or active recombinant caspase), caspase enzymatic activity was measured according to following protocol:

200 l of Caspase Assay Buffer (CAB) containing 20 M fluorescent substrate (Ac-DEVD-amc Ac-YVAD-amc, Ac-VEID-amc; Calbiochem) was added to the activation reaction. The samples were transfered into the wells of a 96 flat-bottom well plate. After incubation in the dark for 30 min at RT, fluorescence was measured at 360/460 nm with a FL500 fluorimeter (Bio-Tek). If a standard-curve of various concentrations (0 up to 5 M in CAB) of aminomethylcoumarin (amc) is measured, the enzyme activity can be calculated and expressed as pikomoles of substrate (amc) hydrolyzed per microgram of protein (in the extract) per minute [pmol g-1 min-1] or just as change of the amc concentration in nanomolar per microgam of protein [nM g-1].

Recipes
Extract Dilution Buffer (DB):

COMPOSITION:
RECIPE for 500 l:

10 mM HEPES (pH 7.0)
5 mM EGTA
50 mM NaCl
2 mM MgCl2
1 mM DTT

supplemented with ATP regeneration system:

2 mM ATP
10 mM phosphocreatine
50 g/ml creatine kinase

100 l of 50 mM HEPES, pH 7.0 incl.
25 mM EGTA
25 l of 1 M KCl
10 l of 100 mM MgCl2
0.5 l of 1 M DTT

-

5 l of 200 mM ATP in water
10 l of 500 mM phosphocreatine in water
10 l of 2.5 mg/ml creatine kinase in KPM buffer

365 l H2O nuclease free

Add DTT and ATP regeneration system always fresh to the buffer, just before use!

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Caspase Assay Buffer (CAB):

COMPOSITION:
RECIPE for 50 ml:

50 mM PIPES
0.1 mM EDTA
10% glycerol
1 mM DTT

838.4 mg PIPES
10 l of 0.5 M EDTA
5 ml glycerol
-

add 40 ml H2O, adjust pH=7.2 using KOH (about 200 l of 1M KOH), then fill up to the final volume of 50 ml.

1mM DTT is always added fresh to the buffer, just before use.

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Lysis Buffer (for DNA isolation from nuclei)

COMPOSITION:
RECIPE for 50 ml:

50 mM Tris-HCl, pH 8.0
10 mM EDTA
0.2 % SDS

5 ml of 0.5 M Tris-HCl, pH 8.2
1 ml of 0.5 M EDTA, pH 8.0
1 ml 10% SDS

add 43 ml H2O

before use, add proteinase K to a concentration of 0.5 mg/ml

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TE Buffer

COMPOSITION:
RECIPE for 50 ml:

50 mM Tris-HCl, pH 8.0
1 mM EDTA

5 ml of 0.5 M Tris-HCl, pH 8.2
100 l of 0.5 M EDTA, pH 8.0

add 44.9 ml H2O

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4% Paraformaldehyde in PBS

RECIPE for 100 ml:

Heat 80 ml H2O to 60C
under stirring add 4g Paraformaldehyde
cover, let stir at 60C, but do not overheat
add 2 drops of 1 N NaOH: solution becomes almost clear, but contains some particles that will not dissolve
add 4 ml of 25x PBS
adjust pH to 7.0 with HCl
bring to final volume with H2O filter solution and store in brown glass bottle at 4C
Solution is good for at least 1 year.

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Activation of apoptotic activity in cytoplasmic extracts and