EMSA

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):

• 10 mM Tris/HCl pH 7.5
• 1 mM EDTA
• 5 mM MgCl
• 50 mM KCl
• 1 mM DTT
• 1x protease inhib. (Complete)
  
  

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

Annealing of oligos

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

Labeling of annealed oligo with ³²P-alpha-dATP with TdT

(Fermentas #EP0161, Terminal deoxynucleotide Transferase)

• 10 µl 5x reaction buffer
• 2.5 µl annealed oligo (10 pmol of 3’ termini = 5 pmol ds-oligo)
• 5 µl ³²P-alpha-dATP (10 µCi/µl > 50 µCi)
• 2 µl TdT (40 u)
• 30.5 µl A.dest. nuclease free

incubate at 37°C for 15 min
(Stop the reaction by heating to 70°C for 10 min).

Incubation of extracts with oligos and native PAGE

(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