What to Consider When Using Phosphoprotein Antibodies in WB?

In the early days, scientists studied unknown signaling pathways using radioactive labeling to track phosphorylation. It worked, but created safety and waste issues. 

Thankfully, the development of phospho-specific antibodies made things easier and safer. However, while these antibodies are useful, interpreting the results still requires caution — they can have their own limitations and should be used carefully.

Phosphoproteins are not very common, and their phosphate groups can be easily lost. It makes them tricky to work with. When using phosphoprotein antibodies in Western blotting, it’s important to handle samples carefully to avoid mistakes.

Be sure to:

• Protect the phosphate groups, which can break off easily.
• Use phosphatase inhibitors, or enzymes might remove the phosphate by accident.
• Use protease inhibitors, or the proteins might break down.
• Handle samples gently, because rough lab work can lower the chances of detecting the phosphoprotein correctly.

To make sure your phosphoprotein stays intact (not dephosphorylated or broken down), follow these steps:

1. Use Inhibitors

Add phosphatase and protease inhibitors to your lysis buffer. These prevent enzymes from removing phosphate groups or breaking down your protein. You can also consider adding kinase inhibitors, depending on your experiment.

2. Work Fast and Keep It Cold

Speed and temperature matter. Process tissues or cells quickly, keep everything on ice, and sonicate to fully break open the cells. This helps preserve the phosphorylation signal.

3. Watch the pH

Even though phosphorylated bonds are usually stable outside normal body pH, pH changes can affect how well antibodies recognize your target. Also, incorrect pH in running or transfer buffers can hurt gel performance and transfer quality.

4. Blocking and Antibody Incubation

Block your membrane with 5% nonfat dry milk in TBS-T (not PBS-T). The milk doesn’t interfere with phosphoprotein detection. However, when using phospho-specific antibodies, it’s better to dilute them in 5% BSA, not milk.

Why not PBS-T? It contains phosphate, which can interfere with phospho-specific antibodies. Use TBS-T instead.

5. Order of Probing

If you’re planning to strip and re-probe the membrane, always probe for the phosphoprotein first. Stripping can damage or remove the phospho-epitope.

6. Incubation Time

Because phosphoproteins are often present in small amounts, it’s best to incubate overnight at 4°C with the primary phospho-antibody for a stronger signal. This is especially true when working with low-abundance targets like those detected by a c-Abl antibody.

7. Multiplexing

If you have the right imaging tools, you can use fluorescent antibodies to detect both the total protein and the phosphoprotein at the same time.

8. Use Controls

Always use a positive control to confirm phosphorylation. You can also use specific treatments or phosphatase treatment as negative controls.

9. Signal Too Low?

Phosphoproteins are usually in low amounts. If you don’t see a signal, try concentrating the protein using immunoprecipitation before running the blot.

Finally

Once you’ve optimized all your steps, you should be able to detect your phosphoprotein — even if it’s tricky — usually one phosphorylation site at a time. 

However, keep in mind that some proteins have multiple phosphorylation sites, which can make things more complex and affect how the signal appears on your blot.