Investigating the role of the JAK/STAT and MAPK ... - UCL Discovery

mM NaCl, 1 mM EDTA, 1 mM EGTA and protease/phosphatase inhibitors as per buffer A)
was added to the pellet and was incubated at for 15 min with shaking. The extract was spun
at 13000 x g for 5 min and the supernatant was stored at -.
2.5.8 Electromobility Shift Assay (EMSA)
EMSA is a method of measuring transcription factor binding in nuclear lysates using
readiolabelled oligonucleotide probes corresponding to the consensus binding site of the
transcription factor of interest. Complimentary oligonucleotides were annealed in STE buffer
(10 mM Tris, 50 mM NaCl, 1 mM EDTA) by heating to for 5 min, then allowing to cool to
room temperature. 100 ng of annealed oligo were labeled with 30 µCi dCTP using 200 U
MMLV reverse transcriptase. 10 µg nuclear extract was added to 500 pg probe, 1 µg
polydIdC and diluted in binding buffer (10 mM HEPES, 50 mM KCL, 50 mM NaCl, 25 mM
KPO4, 5 mM , 2 mM DTT, 1 mM EDTA and 10% glycerol) followed by incubation at rt for
15 min. Samples were electrophoresed on polyacylamide gels, dried for 1hr at and exposed
to autorad film at -.
2.6 Gene Expression Analysis
2.6.1 RNA Extraction
For RNA extraction from cells, 0.5-1 ml Trizol (Invitrogen) was added directly to the tissue
culture dish, tissue was ground to a fine powder under liquid nitrogen and homogenized in 1
ml Trizol using a dounce homogenizer. Samples were incubated at rt for 5 min and 200 µl
chloroform was added per 1 ml Trizol, samples were mixed vigorously and incubated at rt for
3 min. Samples were centrifuged at 12,000 x g for 15 min at . The upper aqueous phase was
transferred to a new tube and RNA was precipitated with 0.5 ml propan-2-ol for 15 min at rt.
Samples were centrifuged at 12,000 x g for 20 min at and the pellet was washed in 1ml 70%
ethanol. RNA was centrifuged at 7,500 x g for 5 min at , residual ethanol was removed and
the pellet was allowed to air dry for 15 min. The pellet was resuspended in 25 µl of
diethylpyrocarbonate (DEPC) treated water and concentration was measured
spectrophotometrically at . A / of ~ 1.8 was considered free of protein contaminants and an /
of ~ 2.0 was considered free of phenol contamination. For qPCR and gene array applications,
RNA purity was examined using the Bioanalyser 2100 (Agilent Technologies, Palo Alto,
CA).
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