This is a cached page for the URL (http://www.meduniwien.ac.at/user/johannes.schmid/EMSA.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
EMSA

EMSA

 

Preparing of cell extracts

 

Cells (e.g. 293 cells) of one 6-well (10 cm2, app. 106 cells): add 100 l/well:

1x EMSA lysis buffer (stock sol.: 5x):

       Lysis of cells by 4 freeze/thaw cycles (-80C/37C incubator): on the plates,

       check breakage by microscopy

       suspend with pipette and transfer to Eppendorf tubes > 1 additional freeze/thaw cycle

       centrifugation: 14 000 rpm, 4C, 15 min > take supernatant: measure vol.: approx.75 l
( > can be frozen at -70C)

       add glycerol to 10% final conc., add KCl to 150 mM (4.2 l 1 M to 75 l sample)

 

Determine protein concentration of the extracts with Bradford reagent:

standards: BSA: 0, 1, 2, 3, 4 g (l) in 96well plates

extracts: 1 l each (or diluted);

+ Biorad Bradford reagens (1:5, 200 l) > measure OD595 in a microtiter plate reader

expected concentration of extracts: 2 3 g/l

 

 

 

Annealing of oligos

 

       equimolar amount of sense and antisense oligo: 400 pmol each (approx. 5 g): 4 l

       20 l 10x Buffer B (Roche)

       A.dest. ad 200 l (172 l)

       heat to 95C (5 min)

       switch off the thermoblock and let cool down to RT

 

concentration: 400 pmol/200 l = 2 pmol/l

 

 

 

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

(Fermentas #EP0161, Terminal deoxynucleotide Transferase)

 

 

incubate at 37C for 15 min
(Stop the reaction by heating to 70C 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 l extract,

-          1 l 1x EMSA lysis buffer (or competitor > 20x molar excess)

-          0.5l 32P-Oligo

> incubated 15 min at RT

+ 1 l 1x EMSA lysis buffer (+ small amount bromphenolblue)

 

-          pipetted into 4 l 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 15C 10 Vh

Step 4.2. 400 V 1.0 mA 2.5W 15C 2 Vh

Step 4.3. 400 V 10.0 mA 2.5W 15C 140 Vh