Western or ELISA – when you think about it, they are really they same assay. You stick some protein down to a support, incubate with antibodies and use a substrate that produces color, light or fluorescence to detect a protein of interest.
Both assays rely on antigen-antibody interactions to specifically detect a target protein out of a complex protein mixture.
So does it matter which technique you use?
It sure does.
Each assay has distinct advantages and differences that determine the information you can glean from your data. And what you need to know about your protein will decide which assay is best for you.
Below are some points to consider when choosing the best assay to use.
Is your protein there?
If you just want to know if a sample contains your favorite protein, then you can use either a Western or ELISA. As long as you know the assay has enough sensitivity to detect your protein, it shouldn’t matter if you run it and blot it or drop it and adsorb it.
You have to analyze more samples than you know what to do with
If you are inundated with samples, then Westerns are not for you! Western blots are more time-consuming and complex than ELISAs and unless you have a team of minions working for you, there is no way you are going to blot hundreds of samples. Already In a microplate format, ELISAs are the assays to use for lots of samples. And if you are really lucky, you will even have an automated plate washer to help you out!
Not many people have minions working for them in the lab. Photo courtesy of Ayca Wilson.
You need more information about your protein and/or sample
ELISAs tell you if your protein is there, and when they are done quantitatively, how much there is. THAT IS IT! You cannot determine the molecular weight of your protein, nor observe size shifts due to posttranslational modifications. You also cannot see degradation products to determine the quality of the sample as a whole. For this type of information, you are going to have to run a gel and blot it.
You need to know how much protein you have
Sometimes just knowing your protein is present is all the information you need, but often you also want to know how much of your protein you have. You can do a quantitative Western, if you do it the right way. But it might be easier to use an ELISA with a standard curve to quantify your protein.
You are developing a medical test
A Western or ELISA can be used in medical diagnostics. Often, ELISAs tend to be the first test used when analyzing a sample because they are quicker, somewhat automated and can test many samples at once. However, ELISAs are prone to false positives, so if an ELISA gives a positive result, a Western blot might still be used to confirm the diagnosis.
Your antibody only recognizes a linear epitope
During ELISA assays, antibodies bind to proteins in solution in their 3-d conformations. When Western blotting, proteins are usually denatured prior to recognition by antibodies. The specificity of your available antibody might dictate the type of assay you can perform.
You are suspicious about the specificity of your antibody
A big drawback to ELISA assays is the fact that you have to trust that your antibody is detecting the right protein. If you are suspicious about the specificity of your antibody, then you need to do a Western blot to make sure the molecular weight of the detected protein is accurate and that no other protein bands are recognized by the antibody.
Time is of the essence
Western blots are more time-consuming than ELISA assays. And as mentioned above, they are not high throughput assays. If you need to analyze your samples quickly, then you might want to consider an ELISA.
Developing your own assay is a drag
If you are going to run a Western blot, then you are almost guaranteed to be going it alone. YOU will be optimizing the assay and determining which blocking and washing buffers to use. YOU will be determining which concentrations of antibodies give the best results and YOU will be deciding which substrate is ideal.
If you want to run an ELISA, then you might be one of the lucky ducks for whom an assay has already been developed and is commercially available. In that case, you will be able to purchase a kit containing a pre-coated microtiter plate and all of the reagents to perform the assay.
A Western or an ELISA give important information about your protein. Make sure you know which assay is the best for you!
Photo courtesy of Jinx!