f)
Technique of Blot Western
Total proteinic extracts are carried out. The cells are washed
twice with the PBS 1X, the base is included in 300ul plug of extraction
(Hepès 20 mm pH 8, NaCl 450 mm, EDTA 0,4 mm and glycerol 25%)
containing inhibiters of proteases (pepstatine, leupeptine, aprotinine). For
the lysis of the cells, three successive baths in nitrogen and with 37°C
are carried out. After centrifugation (15min. with 14000 rpm), the supernatant
corresponding to the total extract is taken, made up in aliquot fractions and
is frozen with - 80°C.
After a denaturation of 10 min. with 100oC, the protein
samples are deposited on a denaturing gel of acrylamide 12%. The migration is
done in 2 stages, first of all to 80 V during 15 min. then with 125 V during
one hour. The proteins are then transferred on a membrane from nitrocellulose
by électrotransfert to 600 my during 60 min. cold. To use the anti-RAR
and anti-RXR antibodies, the membrane is washed twice with PBS 1X then
saturated with milk 5% during 2 hours at ambient temperature. During all the
night, the membrane is incubated with 4oC with the anti-RAR primary education
antibody diluted with the 1/100è and anti-RXR diluted with the
1/100è. Washed 5 times 10 min. with PBS 1X, the membrane Re-are
saturated with milk (2,5%) during 10 min. then incubated with the secondary
antibody during 30 min. (Protein A diluted to the 1/10000è). Then, 5
washings are carried out at the PBS 1X containing 0,01% of Tween.
For the anti-myc antibody, the membrane is saturated with PBS
Tween 20 to 0,1% during 2 hours at ambient temperature. Several washings with
the PBS Tween are made during two min. the membrane is incubated with 4oC all
the night with the anti-myc primary education antibody diluted to the
1/10000è. Washed several times during 5 min. with 500 ml of PBS Tween,
the membrane is incubated with the secondary antibody during one hour
(anti-kappa of mouse diluted to the 1/1500è). Then the membranes are
washed with 1l PBS Tween during at least three times 15min.
Lastly, the membrane is put in the presence of a solution of
detection (ECL, Amersham, Piscataway, NJ, EUA) during one minute. Radiography
is made by exposing a film to variable times (Hyperfilm ECL, Amersham,
Piscataway, NJ, EUA).
G)
Technique of the delay on freezing
The probe DR5, which corresponds to the RARE brief reply of
the promoter of RAR, is marked at its end 5 ' while incubating during 60 min.
with 37oC: 30 ng of the probe, the plug kinase 10X, 3 L of ATPP32, 1 L of T4
polynucléotide kinase (5 units). After having purified the probe marked
on a column (centri.spin-40, hydrated beforehand with 0,5 ml of plug YOU 1X
during 30 min. and centrifuged 2 min. to 700 G at ambient temperature) by
centrifuging it 2 min. to 700 G at ambient temperature, the control of the
specific activity is carried out thanks to a scintillation counter. The probe
doubles bit is elaborate while adding to the marked probe 60 ng of the
complementary bit, the plug kinase 10X and 150 mm of NaCl. The solution is
denatured with 100oC then gradually cooled with 37oC.
The proteinic extracts and the antibodies are incubated 20
min. on the ice with a solution containing 2 ul plug Darnell (KCl 400 mm,
Hepès 200 mm, MgCl2 10 mm, EGTA 1 mm, DTT 5 mm, Ficoll 4%, pH 7,5),
1 ul of poly dI-cd. (1 ug/ul) and 40000 cpm of the radiomarquée probe.
The extracts are then deposited on a polyacrylamide gel 4% and migrated to 200
V during 82 min. freezing is dried in neutral during 120 min with 80°C. An
autoradiography is carried out with an amplifying screen in the presence of a
film Kodak X-Omat AR with- 800C during 12 hours.
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