In this next step, we will transfer our separated proteins out of the gel and
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.