The Invention of Phospho Antibodies Changed How We Study Proteins
Proteins do their jobs based on their shape, so how they’re shaped is key to how they work.
Some proteins act like the framework of a building, giving cells their structure and staying active all the time. Other proteins help control important processes like cell division, and these need to be turned on or off depending on what the cell needs.
One of the main ways cells control protein activity is through a process called phosphorylation. This happens when an enzyme called a kinase adds a small chemical group (called a phosphate) to certain parts of a protein, usually the amino acids serine, threonine, or tyrosine.
Adding this phosphate group changes the shape of the protein. And because shape determines function, this also changes what the protein can do.
This process—turning proteins on or off by changing their shape—is essential to how cells work, and phospho-specific antibodies have become a powerful tool to help scientists study it.
The Early Days Of Using Radiation
At first, scientists had to use radioactive phosphorus (called 32P) to detect phosphorylated proteins. This method was difficult and time-consuming. That’s because thousands of proteins in a cell can get phosphorylated and pick up the radioactive label.
To study just one protein, scientists had to pull it out from all the others using a process called immunoprecipitation.
Even then, they couldn’t tell which exact part of the protein had been modified. This is a big problem because different parts of the protein can have opposite effects—some turn it on, others turn it off.
There were other issues too:
Changes in cell metabolism could affect how much 32P was taken up, making results hard to measure.
Most importantly, it was nearly impossible to study proteins inside living organisms, because labeling with 32P doesn’t work well in live animals or humans.
The Development of Phospho-Specific Antibodies
The story of phospho-specific antibodies began several decades ago, starting with antibodies that recognized a type of phosphorylation on a protein called phosphotyrosine.
The Early Days
In 1981, scientists made the first antibodies that could detect phosphotyrosine by injecting a chemical called benzyl phosphonate into rabbits. These antibodies were very helpful in cancer research, but they had a big limitation:
1. They detected many different proteins, not just one.
2. They also couldn’t tell which specific site on the protein was phosphorylated.
So, while these early tools showed that phosphorylation was happening, they didn’t show exactly where or on which protein it was happening.
Attempts to make antibodies that detect other types of phosphorylation, like phosphoserine or phosphothreonine, mostly didn’t work well.
Trying Whole Proteins
Next, scientists tried using entire phosphorylated proteins to make more specific antibodies. One early success was with a protein called G-substrate, found in brain cells. Researchers made antibodies by injecting phosphorylated G-substrate into rabbits.
But this method often failed for two reasons:
1. Phosphorylated proteins can lose their phosphate groups quickly during the process.
2. Whole proteins have many parts that trigger an immune response, so the body might not make antibodies against the small phosphorylated piece that scientists actually care about.
A Better Way: Using Small Peptides
Eventually, scientists found a better approach. They started using short synthetic peptides—tiny pieces of proteins (15–20 amino acids long) that include the phosphorylated site they want to target.
This method has several advantages:
1. It helps the immune system focus on just the important phosphorylated part.
2. These small peptides are more stable and don’t lose their phosphate groups as easily.
But even this method isn’t perfect. Sometimes, the phosphate still falls off, and the immune system makes antibodies to the non-phosphorylated version too. That’s why researchers have to purify the antibodies carefully.
Purifying the Right Antibodies
To get only the useful phospho-specific antibodies, scientists use a two-step purification process:
1. First, they pass the antibody-containing serum over a filter with the phosphorylated peptide to grab the antibodies that recognize it.
2. Then, they pass it over a second filter with the non-phosphorylated peptide to remove any antibodies that also stick to that version.
This method was developed by one of the co-founders of the work, Andy Czernik, and is now a common approach in labs.
Where Are We Today?
Modern phospho-specific antibodies are much more precise. They bind only to proteins that are phosphorylated at a specific site, so:
- You don’t need to label proteins with radioactive materials like 32P.
- You don’t need complicated separation techniques like immunoprecipitation.
- You can use them in common lab tests like Western blotting (WB) and immunohistochemistry (IHC) to see where and how much phosphorylation is happening.
In IHC, these antibodies can even show which exact cells are active by highlighting where the phosphorylation has occurred.
Understanding Phosphoproteins Has Improved Cancer Treatment
Phospho-specific antibodies have played a huge role in this progress. They’ve made it much easier for scientists to study how proteins are turned on or off by phosphorylation, which has helped in creating many new medicines.
One major group of these medicines is called kinase inhibitors. These are small molecules that block the enzymes (kinases) responsible for adding phosphate groups to proteins. These types of drugs are especially important in cancer treatment.
Here’s why they matter:
- Many cancer cells rely on abnormal phosphorylation signals to grow and divide.
- Kinase inhibitors can block those signals and slow down or stop cancer growth.
- Because they target specific pathways, these drugs often harm cancer cells more than healthy cells, causing fewer side effects.
Today, drugs that affect phosphorylation—especially kinase inhibitors—make up about 25% of all drug research and development.
Beyond Western Blot
Phospho-specific antibodies are commonly used in Western blotting to study phosphorylated proteins. But they’re not used as often in another powerful technique called immunohistochemistry (IHC) or immunofluorescence (IF).
That’s a missed opportunity, because IHC and IF can show exactly where in a cell or tissue a phosphoprotein is active, even down to the subcellular level. This kind of detailed information is very valuable in understanding how proteins work in different parts of the body.
