Phosphorylated proteins play a vital role in cell signaling and function. Detecting them helps scientists and researchers to understand when and how proteins are active in cells.
Western blotting is a common method to find out if proteins are phosphorylated. This technique uses phospho-specific antibodies that can only attach to proteins carrying a phosphate group.
Here we’ll tell you five methods to ensure accurate results and help provide essential details about protein functions in health and disease.
1. Use Phospho-Specific Antibodies
Phospho-specific antibodies are highly specialized tools that bind only to proteins carrying a phosphate group at a precise site—commonly on.
- Serine,
- Threonine,
- Tyrosine residues.
This makes them ideal for detecting when signaling proteins are in their active, phosphorylated state. In Western blot experiments, always choose antibodies that have been validated for both sensitivity and selectivity.
The most reliable phospho-specific antibodies will recognize only the phosphorylated form of the target protein, not the unmodified version.
For optimal results, verify the published validation data and always protect samples from dephosphorylation by adding phosphatase inhibitors during extraction and processing.
2. Perform Multiplex Fluorescent Detection
Multiplex fluorescent western blotting is a method that allows you to visualize both a specific protein and its modified version on the same blot simultaneously. For instance, instead of running two separate experiments, you can use different colored glow sticks to light up two different things in one go.
How it works:
- You use two different antibodies that recognize one of your proteins.
- Each antibody has a unique fluorescent dye attached to it. For example, one antibody might glow red to find the active protein, while the other glows green to find the total amount of that protein.
- When you scan the blot, the different dyes light up in their respective colors. A special camera or scanner captures both colors at once, creating a single image.
3. Use Phosphatase Treatment to Confirm Phosphorylation
To confirm specificity and find out false results, you should treat duplicate blots with a protein phosphatase (such as Λ phosphatase) before examination.
How it works:
- After treatment, true phospho-specific antibody bands should disappear, which confirms detection of the phosphorylated form only.
- Parallel untreated samples serve as positive controls, while phosphatase-treated samples act as negative controls.
- This method is important for antibody validation and helps you in resolving the results of complex samples.
4. Incorporate Phospho-Peptide Competition Assays
Phospho-peptide competition assays are another powerful method to verify phospho-antibody specificity. In this method, the antibody is pre-incubated with either a phosphorylated or non-phosphorylated version of the target peptide before blotting.
How it works:
- If pre-incubation with the phospho-peptide abolishes the signal, it confirms specificity for the phosphorylated form.
- Non-phosphorylated peptides should not compete away the antibody signal.
- This method is perfect for antibody validation by many commercial suppliers. It ensures confidence in results.
5. Use Total Protein as a Loading Control
To obtain accurate results in a Western blot, it is essential to verify that equal amounts of protein were loaded in each lane. Using an antibody that detects the whole quantity of the target protein, both phosphorylated and non-phosphorylated parts, helps to achieve this.
How it works:
- After detecting the phosphorylated protein, you can remove the antibody from the membrane and test it again for the total amount of that protein.
- You can also detect both phosphorylated and total protein at the same time using different colored fluorescent tags.
By comparing the amount of phosphorylated protein to the total protein, you can fix any errors caused by loading different amounts of protein or uneven transfer onto the membrane.
The Bottom Line
Now you know the importance of detecting phosphorylated proteins through Western blotting while studying cell signaling and protein activity. By using these five methods, such as phospho-specific antibodies, multiplex fluorescent detection, phosphatase treatment, peptide competition assays, and total protein controls, researchers can achieve accurate and trustworthy results.