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