Western blot protocol
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- Our simple fast Western blot Protocol
- A list of links to Western blot protocols from other sites
- Links to blot protocols for large proteins, specific cells, tissues
Western Blot Protocol – (Easiest)
Buffers:
TBS: 25 ml of 1 M Tris-7.5, 30 ml of 5 M NaCl, bring volume up to 1000 ml with ddwater
TBS-T: TBS + 0.5 ml of Tween 20
5X SDS-PAGE running buffer: 15.1 g Tris base, 94 g glycine in 900 ml water, then add 50 ml of 10% solution of SDS, and adjust volume to 1000 ml with ddwater
Blocking solution: 5 g nonfat dry milk in 100 ml of PBS
Transfer Buffer: 5.82 g Tris base + 2.93 g glycine + 0.375 g SDS (or 3.75 ml of a 10% solution of SDS) + 200 ml methanol, then bring volume up to 1 liter with ddwater
Prepare cell or tissue extracts using an appropriate protocol.
- Measure total protein in extract by the Bradford method (i like the Pierce kit). Plan on loading approximately 5 ug of protein per lane on the polyacrylamide gel.
- Add an equal volume of SDS sample buffer and boil the sample on a 100 C block for 5 min.
- Apply the sample in about 10 ul volume to an appropriate percentage SDS-PAGE mini gel, along with a visible protein marker (5 ul) and appropriate positive and negative controls. Run gel using 1X SDS running buffer.
- Run gel at 25 mA (constant) for 30 min to 1 hr. (or until blue dye just runs off of the gel)
- Transfer onto PVDF or nitrocellulose using your transfer apparatus, per apparatus instructions.
- After transfer, ensure that all the protein has transferred, as assessed by transfer of the visible protein marker onto the membrane.
- Block in 75 ml of blocking solution overnight at 4 C (alternatively, block at room temp for 1 h on a shaking platform).
- Wash 3 times with TBS-T, each for 10 min.
- Make dilution of primary antibody (usually 2 ul in 10 ml of TBS), and add to membrane, shake on a platform for 1 h at room temp. Make sure that membrane is entirely covered with antibody solution. (alternatively, block at room temp for 1 h on a shaking platform).
- Discard antibody solution and wash with TBS-T 3 times for 10 min each.
- Make dilution of secondary antibody (usually 1 ul in 10 ml), and cover membrane with antibody solution and shake on a platform for 1 h at room temp.
- Discard antibody solution, and wash membrane with TBS-T 4 times for 10 min. each.
- Detection using the ECL/ECL kit of your choice
More Detailed Protocols