Protein detection is a very important for both medicine production and certain clinical diagnosis applications.
One way to perform protein detection is with antibodies that can identify a specific protein.
Protein detection with antibodies is very precise, cost effective, and can be performed relatively quickly.
This article will show how to perform protein detection with antibodies in an ELISA assay.
I will be testing 6 different samples with varying concentrations of the target protein over 3 wells on a plate.
I will also be adding hydrochloric acid (HCl) for a second color change and validation of results.
Background Info (Theory):
There are two types of protein detection using antibodies, Direct and Indirect, but they are both very similar, as can be seen below in the graphic.
Antibodies with a specific enzyme attach to the specific protein, which causes a color change which can be seen if the concentration is high enough.
However for indirect detection, two antibodies are added, where the first one finds the protein and then the other antibody carries that carries the color changing enzyme finds that antibody and attaches, which results in a color change.
For extra validation, another compound can be added to the samples, which in this case is hydrochloric acid (HCl), which changes the color again.
This means that if a sample changes color twice it is almost 100% certain that the sample contains the target protein.
Step 1 (Protein ELISA assay):
The first thing to do is to fill the well plates with the different concentrations of proteins.
Having three or more wells per concentration means that if one of the wells are contaminated, you can use the other wells for data, so fill multiple wells with the same concentrations.
For me, I the lower the number on the left side is, the higher the concentration of target protein.
Step 2 (Color Change):
When you have the well plate, simply drop a few drops of the specific antibody solution for the specific target protein into the wells and observe the color change.
The higher the concentration of the target protein the more red and orange the well will become.
Result and Conclusion:
As you can see in the above picture, there is a stark color change, between well row 6 and well row 1.
This means that ELISA assays are useful and can show if certain proteins are present in a well.
Also if you add hydrochloric acid (HCl) you can get an even better view of how a higher concentration turns the wells from a bright green to an orange color.