Biology Reference
In-Depth Information
3. Methods
Samples should be kept on ice:
3.1. Sample
Preparation
( see Note 10 )
1.
Add complete lysis buffer to the sample in the ratio of 500
μ
L
per 100 mg of tissue or 200
L/4-5 × 10
6
cells. Normally, in a
1.5-mL microcentrifuge tube.
μ
2.
Vortex for 5 min.
3.
Grind or homogenize the sample. We use microcentrifuge pes-
tles optimized for 1.5-mL tubes.
4.
Vortex for 5 min.
5.
Sonicate in ice water for 1 min, let the samples sit for 30 s, and
sonicate for another minute (see Note 11).
6.
Vortex again for 5 min, then centrifuge at 14,000 ×
g
for 20 min
at 4°C.
7.
Collect the supernatant and store at −20°C; also store the pel-
let at −20°C.
Prepare samples as described above (see Subheading
3.1
) in lysis
buffer containing protease inhibitors but no phosphatase inhibi-
tors. Store on ice and move to step 1 immediately:
3.2. PTM-Specifi c
Procedures
3.2.1. Phosphorylation
1.
Take half of each sample and add 1
μ
L of each protein phos-
phatase inhibitor per 100
μ
L of sample.
2.
To the half without phosphatase inhibitors, add 10× reaction
buffer to give a 1× fi nal concentration (i.e., 1
μ
L of 10× buffer
to 9
μ
L of sample). Add 0.5-1 U of alkaline phosphatase/
μ
g
of protein.
3.
Incubate at 37°C for 1 h.
4.
Clean the samples and resuspend them in complete lysis buffer
(see Note 12).
5.
Label both samples as described in Subheading
3.3
below.
1.
Add 1
L of 1 M ascorbic acid stock to 1 mL complete lysis
buffer. Prepare samples as described above in Subheading
3.1
and store samples in lysis buffer with ascorbic acid.
μ
3.2.2. Oxidation
2.
To label oxidized proteins, add a maximum of 60
μ
g of protein
to 7.5
L of lysis buffer to a tube containing 7.5 nM aliquot of
AlexaFluor 488 C
5
-aminooxyacetamide (
5
).
μ
3.
Vortex and “quick-spin” to pool the sample in the bottom of
the tube (see Note 13).
4.
Incubate at room temperature, protected from light, for 2 h.
Then transfer and keep at 4°C overnight.
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