EMSA
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Preparing of cell extracts
Cells (e.g. 293 cells) of one 6-well (10 cm2, app. 106 cells): add 100 µl/well :
1x EMSA lysis buffer (stock sol.: 5x ):
Ø Lysis of cells by 4 freeze/thaw cycles (-80°C/37°C incubator): on the plates,
Ø check breakage by microscopy
Ø suspend with pipette and transfer to Eppendorf tubes > 1 additional freeze/thaw cycle
Ø centrifugation: 14 000 rpm, 4°C, 15 min > take supernatant: measure vol.: approx.75 µl
( > can be frozen at -70°C)
Ø add glycerol to 10% final conc., add KCl to 150 mM (4.2 µl 1 M to 75 µl sample)
Determine protein concentration of the extracts with Bradford reagent :
standards : BSA: 0, 1, 2, 3, 4 µg (µl) in 96well plates
extracts : 1 µl each (or diluted);
+ Biorad Bradford reagens (1:5, 200 µl) > measure OD595 in a microtiter plate reader
expected concentration of extracts: 2 � 3 µg/µl
Ø equimolar amount of sense and antisense oligo : 400 pmol each (approx. 5 µg): 4 µl
Ø 20 µl 10x Buffer B (Roche)
Ø A.dest . ad 200 µl (172 µl)
Ø heat to 95°C (5 min)
Ø switch off the thermoblock and let cool down to RT
concentration : 400 pmol/200 µl = 2 pmol /µl
(Fermentas #EP0161, Terminal deoxynucleotide Transferase )
incubate at 37°C for 15 min
(Stop the reaction by heating to 70°C for 10 min).
(Electrophoresis on PhastSystem , GE Healthcare, formerly Pharmacia-Amersham : see instruction manual of the manufacturer )
- 2 µl extract ,
- 1 µl 1x EMSA lysis buffer (or competitor > 20x molar excess )
- 0.5µl 32P-Oligo
> incubated 15 min at RT
+ 1 µl 1x EMSA lysis buffer (+ small amount bromphenolblue )
- pipetted into 4 µl sample combs of the PhastSystem
- run samples on 12.5% homogenous Phastgels using native buffer strips
Sample Appl . down at 4.2. 0Vh
Sample Appl . up at 4.2 2Vh
Step 4.1. 400 V 10.0 mA 2.5W 15°C 10 Vh
Step 4.2. 400 V 1.0 mA 2.5W 15°C 2 Vh
Step 4.3. 400 V 10.0 mA 2.5W 15°C 140 Vh
