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        EMSA

        互联网

        1956

         

        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

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