TNT reaction (all the handling is on ice)
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rabbit reticulocyte lysate
Reaction buffer
T7 RNA polymerase
1mM amino acid mix(-Met)
35
S-Met
RNase Inhibitor
DNA template (with T7 promoter)
H2O
12.5ul
1ul
1ul
1ul
2ul
1ul
3ug
depends on DNA volume
Total ~30ul
 incubate @30C for 90min. After incubation, store the product @-80C
GST-pulldown Assay
Binding buffer:
HEGN buffer (20mM HEPES, pH 7.9, 1 mM EDTA, 10% glycerol, 0.15 M KCl, 0.05% NP40) + DTT,
PMSF, proteinase inhibitors
Washing buffer:
HEGN buffer with high concentrated salt and detergent
(20mM HEPES, pH 7.9, 1 mM EDTA, 10% glycerol, 250m M KCl, 0.1% NP40) + DTT, PMSF, proteinase
inhibitors
1) Vortex GST-proteins beads first before taking them out)
GST
(~1ug)
GST- fusion protein
TNT protein
12ul
TNT protein
Binding buffer 200ul
Binding buffer
(~1ug)
(12ul)
200ul
Rest of TNT protein can be used for input with 2X sample buffer
2) Rotating @RT, 40min~1hr for binding.
3) 8000rpm @4C for 1min
4) Remove all supernatant and wash w/ 300ul of washing buffer if it is hard to see the bead, add
10~20ul of glutathione beads more.
5) Rotating @RT for 5min. Repeat washing 3 times.
6) After final washing, spin @8000rpm for 1min, remove supernatant and add 30ul of 2X sample
buffer.
7) Run SDS-PAGE Gel (~200V)
8) After running,
 Fixing (50% methanol, 10% acetic acid, 40% water) for 10min @RT with shaking gently,
 Remove solution
 Enhancing buffer for 15min @RT by shaking gently
 Remove or recycle the solution
 Ice-cold water for 15min @RT by shaking gently
 Dry gel and expose to Film