STEWART LABORATORY
TRAP ASSAY
PREPARE LYSATE
Ø
Lyse cells for 20-30 min
on ice in CHAPS lysis buffer
Ø
Spin cells at
4°C for 20 min
Ø
Save supernatant
in a new RNase-free tube
Ø
Quantify protein
concentration using the BioRad kit. Be sure to have appropriate blanks and
concentration controls
Ø
Dilute a small portion
of the sample to 50 ng/μL. Return the undiluted sample to -70°C for
future use
Ø
Take one quarter
of the diluted sample and boil for 5 min and crash on ice. This will serve as your heat inactivated
control
END LABEL TS OLIGO
(this makes enough oligo for 10 reactions):
Ø
Mix:
§
2.5 μL γ-32P-ATP (3000 Ci/mmol)
§
2 μL 10X buffer
§
5.2 μL TS primer
§
9.8 μL ddH2O
§
0.5 μL T4 kinase
Ø
Heat to 37°C for
20 min
Ø
Heat to 85°C for
5 min
SET-UP TRAP REACTION
Set-up
the following 50 μL reaction (this is per
reaction so when performing multiply reactions prepare a supermix). Each sample should have a minimum of two
conditions:
the sample and the heat inactivated control.
You can also set up a dilution series if you are attempting to quantify
the activity
Ø
Mix:
§
5 μL 10X buffer
§
2 μL Primer
§
2 μL labeled TS primer
§
1 μL dNTPs (at 2.5 mM each)
§
0.4 μL Taq
§
37.6 μL ddH2O
§
2 μL prepared extract
RUN REACTION
Set up PCR program:
Ø
1 cycle
30 min at 30°C
Ø
27 cycles
94°C for 30 sec
60°C for 30 sec
RUN GEL
Ø
12.5% acrylamide gel (0.4mm) with 0.5X TBE buffer
Ø
Add 10 μL of 6X loading dye and run 6 μL
Ø
Run gel at about
1000-1200 volts until the first dye runs off and the second dye is about 3/4
the way into the gel
IMPORTANT BUFFERS:
Ø
1X CHAPS
(3-[(Cholamidopropyl)dimethylammonio]-1-propanesulfonate) buffer (All Rnase-free)
·
Mix:
¨
10 mM Tris, pH 7.5
¨
1 mM MgCl2
¨
1 mM EGTA
¨
0.1 mM benzamidine
¨
5 mM 2-mercaptoethanol
¨
0.5%
CHAPS
¨
10%
glycerol
·
Immediately
before lysing cells add 5 mM
-mercaptoethanol (stock from Sigma is at 14.3 M so
add 0.5 μL to 1.4 mL
of the buffer)
Ø
10X TRAP BUFFER
·
Mix:
¨
200 mM Tris, pH 8.3
¨
15 mM MgCl2
¨
630 mM KCl
¨
0.5% Tween 20
¨
10 mM EGTA
¨
0.1%
BSA
Ø
50X dNTPs
·
2.5 mM of each dNTP
OLIGOS:
(Oligos
ordered purified in solution)
Ø
ACX: GCGCGGCTTACCCTTACCCTTACCCTAACC
Ø
TSNT: AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT
Ø
NT: ATCGCTTCTCGGCCTTTT
Ø
TS: AATCCGTCGAGCAGAGTT
CONCENTRATIONS FOR
USE:
Ø
Primer mix II:
§
ACX
0.1 μg/sample
§
NT
0.01 μg/sample
§
TSNT
0.01 amol/sample
Ø
Primer mix III:
§
ACX
0.1 μg/sample
§
NT
0.1 ng/sample
§
TSNT
0.01 amol/sample