Method: Southern Transfer with Immobilon-N

C. Helms

Principle:

Refer to Southern transfer with Zetabind membrane. Immobilon- N is a non-nylon membrane that can be used very much like Zetabind in Southern transfers. Because it is extremely hydrophobic when dry, the membrane must first be prewetted in alcohol prior to use.

Time required:

2 hours to set up the transfer, 2 hours the next day for washing and baking.

Special reagents:

• Immobilon-N is purchased from the Millipore Corp.

Procedure:

{Run the gel as usual, stain and photograph}

Day 1

Pretreatment:

1. Denature the gel for 30 minutes in 0.2M NaOH, 0.6M NaCl with agitation.
2. Rinse with deionized water.
3. Neutralize the gel for 30 minutes in 0.5M Tris-HCl pH 7.5, 1.5M NaCl with agitation.

Transfer:

1. Cut the membrane to be slightly larger than the gel. Label the membrane using a Dry Erase marker. Be sure to mark the bottom of the membrane 'Immobilon-N'. Wear gloves when handling the membrane, and protect it from dirty surfaces by working on a clean piece of blotting paper.
2. Prewet the membrane in freshly prepared 70% ethanol, then place the membrane in deionized water. Rinse briefly, then place in 10X SSC (the transfer buffer). The membrane becomes slightly translucent and feels thicker than nylon membranes. Allow the membrane to equilibrate for 15 minutes.
3. The gel is placed on a 1-2 inch high stack of blot blocks (S &S or BRL) topped with 1 piece of 3MM filter paper, all thoroughly saturated with 10X SSC. The transfer tray should be filled with 10X SSC to within a quarter-inch of the top blot block.
4. Lay the membrane over the gel surface and gently roll a test tube over the surface to remove air bubbles caught between the gel and the membrane.
5. Top the membrane with one or two prewetted 3MM pieces cut to the size of the gel, then place 6-10 dry blot blocks on top, followed by stacks of paper towels (1-2 inches). If the blot blocks and paper towels are not cut to the size of the gel, arrange a barrier (use, e. g. parafilm) around the gel edges to prevent the 10X SSC buffer from bypassing the gel. Top the paper towels with a flat plate and a 500 g weight. (Transfer should take a minimum of 6 hours).

Day 2

Washes:

1. After transfer, soak the membrane in 2X SSC for 15 minutes, then allow to air dry thoroughly before baking 1-2 hours in an 80 degrees C vacuum oven
2. After baking, immerse the membrane in fresh 70% ethanol to rehydrate, then rinse in deionized water. The membrane can now be rinsed at room temperature in postwash II (0.1X SSC, 0.2% SDS. The membrane can be stored in a sealed bag or prehybridized as usual. The membrane can be hybridized and stripped of probe using the same procedures as for nylon membranes.

   NOTE: If at any time the Immobilon membrane becomes dry, it must be prewetted again in 70% ethanol before using. The normal blotting after the hybridization washes should leave enough moisture in the blots so that the membrane need not be prewetted routinely. A dry Immobilon membrane appears like nylon (opaque white), while the slightly damp membrane is thicker and has a yellowish tinge.

References: None