Normalizing your Western blot is critical to obtaining accurate quantitative measurements. But it can be tricky. Perhaps you are discovering that housekeeping genes are not as neat and tidy as you would like? Then you might like to try total protein normalization.
Total protein normalization – what is it?
Total protein normalization (TPN) is normalizing the signal from your protein of interest to the signal from all the other proteins in the same lane. It is a great way to measure the abundance of your protein without relying on housekeeping genes.
TPN has several advantages:
- It is more quantitative because you normalize the signal to many proteins
- You don’t have to find the right housekeeping gene for your experiment
- You can easily detect everything on one blot
- It is more compatible with lower abundant proteins
- Depending on the technique, no special equipment is required
- Stain-free technology can also be used to visualize transfer efficiency
But TPN does come with some disadvantages:
- Some stains cannot be used prior to immunodetection
- Fluorescent stains and stain-free technologies require equipment for visualization
- Some stains fade quickly, are difficult to photograph or require longer incubation times
Is staining for you?
One way to visualize all of the proteins on your blot is to use a protein stain.
Some, like AdvanStain Ponceau, are transient stains that can be used in less than 10 minutes to quickly check the proteins prior to immunodetection. The pink-red stain is easily washed off and fades quickly, so it can be difficult to capture a good image. Some might say it is a great stain for a quick visual inspection of your blot after transfer.
Fluorescent stains are permanent stains that can also be used prior to immunodetection. Most, like AdvanStain Scarlet, are highly sensitive and can be imaged on any fluorescence imaging system. Keep the time in mind though – fluorescent staining procedures can take a bit longer than other stains.
Other stains, such as amido black and colloidal gold, need to be used after antibody detection, but require no special equipment for visualization.
When is a total protein stain not a stain? When it uses stain-free technology!
Stain-free technology uses a trihalo compound that is incorporated into the gel where it binds to proteins during eletrophoresis. When a little UV light is shined on the gel, the compound reacts with tryptophan residues making them fluoresce. Then you just need to take a picture of all the lovely proteins lighting up.
The benefits to stain-free include:
- Ability to visualize proteins at all stages: in the gel after electrophoresis and on the blot after transfer
- No interference with downstream immunodetection
- You can purchase pre-cast gels with the compound already incorporated or make them yourself
As you can see, there are many alternatives to using a housekeeping gene for normalization. Which is your favorite?
Photo courtesy of loa bacon.