There is this old contraption sitting high on the shelf in the lab gathering dust. You’ve never seen anyone use it and you wonder if it is a useless relic or if it is a long-lost piece of equipment that could make your lab life infinitely easier.
You pull it down and discover that it is a dot blotter, otherwise known as a microfiltration unit. It’s an intriguing device, allowing one to spot an army of samples in a beautifully ordered array across a membrane. You vaguely recall using one to look at RNA levels when you were a grad student, but you don’t know if it has any use in your current protein-centric lab.
It turns out, there are may things you can do with some protein and a dot blotter:
Titration of antibodies
Dot blotters are well suited to help you titrate antibodies. Because wells are delineated and physically separated from one another, multiple samples containing different concentrations of primary and/or secondary antibodies can be easily processed simultaneously.
Optimizing blotting conditions
In addition to the titration of antibodies, a dot blotter is ideal for optimizing blocking conditions and washing steps. Each well can test a different condition.
Finding your protein
A dot blotter can be used to help you follow a protein through protein purification steps. Samples from each step in the purification process can be saved and analyzed using a dot blotter to help you determine the elution profile of your protein.
Quantitation of protein in crude samples
If you have purified protein as a standard, you can use a dot blotter to set up a standard curve and then quantitate protein levels in crude samples.
With it’s ability to analyze a large number of samples at one time, the dot blotter is ideally suited for screening multiple samples. Detection of your protein of interest in blood samples, for example, can be easily determined.
Limit of detection
If you have a protein standard that you can titrate, a dot blotter can be used to find the limit of detection of your primary antibody.
What a dot blotter can’t do
A dot blotter can’t solve all of your protein assay needs. And in fact, in some cases, a dot blotter could lead to a false interpretation of results. Since proteins are not electrophoretically separated by size prior to transfer to the membrane, nothing can be determined about the integrity or size of your protein. When using a dot-blotter you cannot:
- Determine if your protein is intact
- Find out if your protein has the correct apparent molecular mass
- Look for post-translational modifications that alter the molecular weight of your protein
- Easily determine if non-specific binding to another protein is occurring
The only thing you can say for sure, is that the dots contain an epitope that is recognized by your antibody. This could be as small as 8-12 amino acids!
However, if you can reasonably assume your protein is intact and you are familiar with the properties of your protein and the limits of the assay, then dust the dot blotter off and put it to work.
Photo courtesy of katsrcool (Kool Cats Photography).