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  • Western Blotting Transfer Protocol (Wet Transfer To Membranes)

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    naveenmi2
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    The document provides instructions for performing a Western Blot transfer using a wet transfer method. Key steps include: 1. Preparing the gel by removing stacking gel and incubating in transfer buffer. 2. Preparing membranes like nitrocellulose by pre-wetting in water and transfer buffer. 3. Assembling the transfer sandwich in a cassette with layers consisting of sponge, filter papers, gel, membrane, and filter papers. 4. Running the transfer in a tank at 25V for 2.5 hours in a transfer buffer solution.

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    Wet Transfer

    Original Title

    313237 Wet Transfer

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    The document provides instructions for performing a Western Blot transfer using a wet transfer method. Key steps include: 1. Preparing the gel by removing stacking gel and incubating in transfer buffer. 2. Preparing membranes like nitrocellulose by pre-wetting in water and transfer buffer. 3. Assembling the transfer sandwich in a cassette with layers consisting of sponge, filter papers, gel, membrane, and filter papers. 4. Running the transfer in a tank at 25V for 2.5 hours in a transfer buffer solution.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    11 views3 pages

    Western Blotting Transfer Protocol (Wet Transfer To Membranes)

    Uploaded by

    naveenmi2
    The document provides instructions for performing a Western Blot transfer using a wet transfer method. Key steps include: 1. Preparing the gel by removing stacking gel and incubating in transfer buffer. 2. Preparing membranes like nitrocellulose by pre-wetting in water and transfer buffer. 3. Assembling the transfer sandwich in a cassette with layers consisting of sponge, filter papers, gel, membrane, and filter papers. 4. Running the transfer in a tank at 25V for 2.5 hours in a transfer buffer solution.

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    © All Rights Reserved
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    of 3

    Western Blotting Transfer Protocol (Wet transfer to

    membranes)
    Gel preparation
    After the electrophoresis run
    - Disassembly the gel and open the gel cassette
    - Truncate the stacking and front of the gel
    - Place the gel in pre-chilled transfer buffer for 10-15 minutes

    Membrane preparations
    - Cut the desired membranes 8 cm X 9 cm
    - Pre wet blotting paper in pre-chilled transfer buffer
    - Pre- wet Nitro Cellulose (NC) membranes in ultra pure water for 20 seconds followed
    by 5 minutes in pre-chilled transfer buffer
    - Pre-wet the PVDF membrane first in methanol for 20 seconds, then quickly in ultra
    pure water for 20 seconds and finally in cold transfer buffer for at least 5 minutes

    Preparing the Tank transfer


    - Fill the transfer unit with transfer buffer, the buffer level should be between min and
    max and according of the apparatus manufacturer.
    - Place the opened cassette with the anode side (+) at the bottom of the pre-filled tray
    with at least 3 cm of transfer buffer.
    - Build the stack on the anode side, according to Figure 1.
    - The sandwich can be prepared at the opposite direction.

    -
    Layer 6 sponge

    Layer 5: Blotting paper

    Layer 5: Blotting paper

    Layer 4: Desired Gel

    Layer 3: NC or PVDF Membrane

    Layer 2: Blotting paper

    Layer 2: Blotting paper

    Layer 1 Sponge

    +
    Figure 1: Schematic picture of the stack (i.e. sandwich)

    - Layer 1: Place a 3 mm-thick foam sponge and press gently until the air is expelled
    - Wet the blotting filter in transfer buffer
    - Layer 2: Place the two pre-wetted blotting papers on the sponge and press gently
    until the air is expelled.
    - Layer 3: Place the desired pre-wetted membranes on layer 2 and remove all air-
    bubbles!
    - Equilibrate the desired gel in cold transfer buffer for 15 minutes
    - Layer 4: Place the gel on the membrane
    - Layer 5: Cover the gel with two pre-wetted sheets of blotting paper
    - Layer 6: Finally place a sponge of 3 mm and again press gently to expel trapped air
    - Close the cassette and press lightly to lock the tabs
    - Place the cassette into the tank.
    -
    Running conditions Transfer/Tank
    - GeBaGel: Transfer at 25 V (maximum 400 mA) and at 4°C with stirring for 2.5 hours

    Transfer buffer (TF), 25 mM Tris, 192 mM Glycine, 20 % methanol


    Prepare freshly.

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