into a solid membrane or blot.

This is based upon the same principal as the previous step in which an electric

field is charged to remove the negative proteins towards a positive

electrode.

Transfer can occur under wet or semi-dry conditions. Here we will demonstrate the

traditional wet transfer method. Start by removing the gel from its cassette

cutting the top portion containing the wells.

Notch the top left corner to indicated gel orientation.

Float the gel in transfer buffer while preparing the transfer sandwich.

To make the transfer sandwich you will need a cassette,

sponge, filter paper,

the gel

gel and your choice of either PVDF

or nitrocellulous membrane.

PVDF must first be activated by soaking the membrane in ethanol for

two minutes.

But other than this the PVDF or choice of nitrocellulous

membrane is a personal preference.

Notch the top left corner to indicate blot orientation

and incubate membranes in transfer buffer for 10 minutes.

Create a stack by placing the following components from the black

negative cathode to red positive anode:

sponge,

filter paper, gel,

membrane.

(Be careful not to touch the gel or membrane with your bare hands and

use clean tweezers or spatula instead.

Touching the membrane during any phase can contaminate the blot and lead to

excessive background signal.)

filter paper

and sponge.

Use a clean roller with each layer to gently roll out any bubbles that may be

present since bubbles will inhibit efficient protein transfer.

Lock the cassette and place it in the apparatus containing cold

transfer buffer

ensuring that the cassette is properly positioned from negative to positive.

In order to prevent heat buildup, it is beneficial to transfer with a cold

pack in the apparatus or in a cold room with the spinner bar placed at the

bottom of the chamber.

Close the chamber and connect to a power supply.

Perform the transfer according to the manufacturer's instructions which is

normally 100 volts for thirty to one hundred and twenty minutes.