Wish all your Westerns look like this one?
Western blot .org
Western blots are a mainstay of any life scientist’s existence. Like most scientists, the two of us have spent countless days, weeks, and months of our lives trying to get a beautiful western blot for publication. Enough bad western blotting!
WESTERN BLOT EXPERTS WE NEED YOUR HELP! If you have a great troubleshooting protocol please share it via the Get Help Page.
This site is dedicated to helping scientists simplify and troubleshoot Western blots. It is always under construction, so pardon the dust and happy western blotting.
On it you will find specific articles about techniques that work including:
- Getting started with Western blots
- Troubleshooting Western blots
- Best antibodies for Western blot detection
- Dotblots vs western blotting
- Western blotting analysis
- Tissue blots
- Cell Lysis for Western blotting
- Large protein Western blots
Do you have a specific problem with your Western blots that we haven’t addressed? Ask it!
The Basics
Plain and simple a Western blot is the most common way to detect a specific protein or group of proteins in a biological sample. Proteins are detected with specific primary antibodies, which can be used to estimate the relative amounts of the protein in the sample.
The principle Western blot protocol is as follows:
- Samples are prepared in homogenization buffer typically containing detergents to solubilize proteins from cells and tissues
- Samples are run on a SDS-PAGE gel to separate proteins based on size and charge
- Separated proteins or “bands” are transferred to a membrane
- Membranes are then “blocked” by incubating with solution containing non-specific proteins
- Specific antibodies are added in incubation buffer which bind to the protein bands of interest
- Secondary antibodies conjugated to enzymes are added to recognize the primary antibody bound to the specific protein bands
- Finally, enzyme substrates are added and the bands are finally visualized
Sounds simple right? Guess again. We estimate that time needed for a scientist to achieve a “publishable” Western blot is 6 months from starting the project. That’s SIX MONTHS for your western blotting madness.
Western blotting is tough business
Why 6 months? Because there are so many steps and factors to troubleshoot. What blocking solution to use for western blotting? Is the primary antibody any good? What detection method is best? Concentration? Incubation Time? Incubation Temperature?
If you’ve gotten this far its time to get specific, what do you want to fix about your Western blots? The links on the Nav Bar and and sidebar will take you to specific articles that can help your Western blotting.