Western Blot Protocol

This western blot protocol should be used as a guide for each researcher to build their own protocol, as each reagent will need to be optimized for use in the particular species, tissue type and application combination.

Block

1. Use 5% skim milk powder in 1X TBST as the blocking buffer.
2. Use excess blocking buffer so that the blot is fully covered.
3. Block for 1 hour at room temperature with shaking.

Wash

1. 3 x 4 minutes in 1X TBST and then 3 x 4 minutes in 1X TBS.
2. Make sure there is an excess of wash buffer in the box at all times.

Primary Antibody Incubation

1. Dilute the primary antibody in TBST.
2. Incubate at room temperature on the shaker for 2 hours.
3. Occasionally check the blots to make sure they are well covered in the antibody solution.

Wash

1. 3 x 4 minutes in 1X TBST and then 3 x 4 minutes in 1X TBS.
2. Make sure there is an excess of wash buffer in the box at all times.

Secondary Antibody Incubation

1. Prepare your secondary antibody in 1X TBST (concentration will depend on the antibody being used).
2. Incubate at room temperature on the shaker for 1 hour.
3. Occasionally check the blots to make sure they are well covered in the antibody solution.

Wash

1. 3 x 4 minutes in 1X TBST and then 3 x 4 minutes in 1 X TBS.
2. Make sure there is an excess of wash buffer in the box at all times

Detection

The volume of ECL detection reagent used for a blot depends on the size of the blot. As a general rule, you just need pipette enough solution over the blot so that it is covered.

1. Mix solution A and B in a 1:1 ratio.
2. Pick up the blot with tweezers and gently tap an edge against a kim wipe to drain excess Tris from the blot.
3. Place the blot in a new (clean) box and pipette the detection reagent over the blot so that it is evenly covered.
4. Incubate the blot (in the dark) for 5 minutes.
5. Using tweezers pick up the blot and drain excess reagent using a kim wipe.
6. Image the blot.