In the early days of studying how cells send signals, scientists used radioactive labels (like radiolabeled phosphate). It was done to track protein phosphorylation. While effective, it created radioactive waste and was tricky to manage.
Then came a game-changer: phospho antibodies. These antibodies can detect when a protein has a phosphate group added to it.
They made the process easier, faster, and safer—but interpreting results with them still requires care.
What Is Protein Phosphorylation?
Phosphorylation is like a switch that turns proteins on or off to control signals inside cells. The process is controlled by:
- Kinases, which add phosphate groups
- Phosphatases, which remove them
Usually, the switching happens on specific parts of proteins, like –
- serine (Ser),
- threonine (Thr),
- histidine (His),
- aspartate (Asp), and later,
- tyrosine (Tyr).
These modifications help pass messages inside cells and are involved in nearly every major biological process, including:
- Cell growth and division
- Metabolism
- Nervous system activity
- Immune response
- Cancer development
How Do These Antibodies Help?
Scientists need to know whether a protein is phosphorylated if they want to know what’s happening inside a cell. Phospho antibodies can help detect everything. Further, they can –
- Recognize general phosphorylation, like on any tyrosine
- Detect specific patterns or “motifs” around the phosphorylated site
- Target a very specific phosphorylated region on a single protein
For most researchers studying signaling pathways, these highly specific antibodies are the best choice when available.
What Are The Things To Watch Out For in the Lab?
When using phospho-specific antibodies in techniques like western blotting, you need to be careful:
- Phosphorylation is rare, so signals can be weak
- The phospho bond is delicate and can break easily
- Phosphatases (enzymes in your sample) might remove the phosphate group if you don’t inhibit them
Poor lab practices, like skipping inhibitors or not keeping proteins cold, can lead to:
- Loss of phosphorylation
- Protein breakdown
- Weaker antibody signals
Important Considerations
1. Protect the Phosphoproteins
Add inhibitors to your lysis buffer:
- Use protease and phosphatase inhibitors to prevent proteins and phosphate groups from breaking down.
- You may also consider kinase inhibitors to freeze the phosphorylation state.
Work quickly and keep everything cold during tissue/sample preparation. Lastly, use sonication to fully break open the cells and ensure complete lysis.
2. Mind the pH
Phospho bonds are generally stable at non-physiological pH, but:
- Some antibodies may not work well if pH changes too much.
- Improper pH in buffers can mess up gel separation or transfer to membranes.
3. Block the Membrane Properly
Use 5% nonfat dry milk in TBS + 0.1% Tween-20 to block the blot.
| Milk works fine; pasteurization kills any alkaline phosphatase that could interfere. |
However, for phospho-specific antibodies, dilute and incubate them in 5% BSA instead of milk.
4. Choose Your Buffers Wisely
Use TBS-T, not PBS-T.
PBS contains phosphate, which can interfere with phospho-antibody binding.
5. Plan for Reprobing
Always probe for the phosphorylated protein first.
| Stripping the blot (to reuse it) can cause loss of the phospho signal. |
Afterward, you can probe for total protein as a control (to check loading/transfer).
6. Consider Multiplexing
If you have the equipment, try fluorescent multiplexing.
This lets you measure phosphorylated and total protein at the same time.
7. Use Proper Controls
- Use a positive control sample known to be phosphorylated.
- Use activators/inhibitors to increase or block phosphorylation as needed.
- Phosphatase treatment can be used as a negative control to show that your antibody is specific.
- Peptide competition assays can also confirm specificity (using phosphorylated vs. non-phosphorylated peptides).
8. Low Signal? Enrich the Target
Phosphorylated proteins are often in low amounts.
If you get no signal, try immunoprecipitation to concentrate the protein before running the blot.
9. One Protein, Multiple Sites
Some proteins have more than one phosphorylation site.
You might see multiple bands or unexpected results, depending on which site the antibody recognizes.
Final Tip
If you optimize all the steps, you should be able to detect that elusive phosphoprotein—one phosphorylation site at a time!
