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
- Use 5% skim milk powder in 1X TBST as the blocking buffer.
- Use excess blocking buffer so that the blot is fully covered.
- Block for 1 hour at room temperature with shaking.
Wash
- 3 x 4 minutes in 1X TBST and then 3 x 4 minutes in 1X TBS.
- Make sure there is an excess of wash buffer in the box at all times.
Primary Antibody Incubation
- Dilute the primary antibody in TBST.
- Incubate at room temperature on the shaker for 2 hours.
- Occasionally check the blots to make sure they are well covered in the antibody solution.
Wash
- 3 x 4 minutes in 1X TBST and then 3 x 4 minutes in 1X TBS.
- Make sure there is an excess of wash buffer in the box at all times.
Secondary Antibody Incubation
- Prepare your secondary antibody in 1X TBST (concentration will depend on the antibody being used).
- Incubate at room temperature on the shaker for 1 hour.
- Occasionally check the blots to make sure they are well covered in the antibody solution.
Wash
- 3 x 4 minutes in 1X TBST and then 3 x 4 minutes in 1 X TBS.
- 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.
- Mix solution A and B in a 1:1 ratio.
- Pick up the blot with tweezers and gently tap an edge against a kim wipe to drain excess Tris from the blot.
- Place the blot in a new (clean) box and pipette the detection reagent over the blot so that it is evenly covered.
- Incubate the blot (in the dark) for 5 minutes.
- Using tweezers pick up the blot and drain excess reagent using a kim wipe.
- Image the blot.