Protein phosphorylation is a post-translational modification in which the function and activity of a protein is significantly altered by adding a phosphate group. The phosphate group attaches to hydroxyl group (-OH), a chemical functional group present on the side chains of typically serine, threonine, or tyrosine amino acids. Researchers rely on phospho-specific antibodies to precisely interrogate these modifications in the protein.
Phospho-specific antibodies are immunoreagents that bind to an amino acid sequence that is phosphorylated in the protein.
Producing high-quality phospho-specific antibodies is a complex, multi-step process that includes:
Antigen design
Peptide synthesis
Carrier conjugation
Immunization
Affinity purification
Rigorous validation
Antigen Design and Peptide Selection
The production begins with the selection of a short chain containing 10-15 amino acids with one or more of the amino acids phosphorylated. The phosphorylated amino acids are positioned in the center of the amino acid chain for optimal accessibility for binding.
Amino acids immediately next to the phosphorylated amino acids should be the native sequence of the protein. The process uses the following two versions of the peptide for production and testing.
A phosphorylated peptide contains phosphorylated serine, threonine, or tyrosine residue.
A non-phosphorylated control peptide is a matching version but lacks a phosphate group at the same position.
Peptide Synthesis and Quality Control
The production requires peptides with crude yield exceeding 85% and at least 95% purity. Both versions of peptide (phospho‑ and non‑phospho) require mass spectrometry confirmation to ensure quality control and specificity.
Verifying correct modifications at the phosphorylation site is crucial to ensure correct binding. It is important to verify unintended side reactions to maintain peptide structure and integrity.
Each peptide is dissolved in a PBS or a bicarbonate buffer at pH 8.3. This is followed by peptide concentration measurement by amino acid analysis or UV absorbance if aromatic residues are present. High-purity, quality-controlled peptides in the final, stable form are known as lyophilized peptides.
Lyophilized peptides are divided into aliquots to preserve the integrity and stability. These peptides are then stored at –20 °C to prevent degradation, protect against moisture and preserve integrity for subsequent steps, especially the conjugation step.
Carrier Protein Conjugation
As small peptides are unreliable to induce the intended immune response, they are chemically linked to a keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) to boost immunogenicity.
Peptide synthesis uses C-terminal cysteine which serves as a conjugation handle. The thiol group, a chemical functional group present on peptides, reacts with a maleimide‑activated carrier to ensure site-specific attachment. This reaction takes place under mild conditions (pH6.5-7.7).
Each carrier molecule carries 10 to 20 peptides, a sufficient number to create the intended binding sites without overloading the protein. Excess free peptide is removed by dialysis or spin-column filtration. Subsequently, conjugation efficiency is verified using methods such as SDS-PAGE, and if possible, UV-vis spectrophotometry to quantify peptide density.
Immunization Strategy
The selection of the host species depends on the downstream applications and desired yield.
Rabbits offer high-titer polyclonal sera.
Mice offer potential for high hybridoma generation.
Goats offer large-scale antibody production.
The phospho-peptide–carrier conjugate emulsification in complete Freund's adjuvant initiates prime immunization. This is followed by incomplete Freund’s adjuvant for subsequent booster injections. 3 to 4 booster injections are administered at 2–4-week intervals.
Test bleeds are collected after each boost to:
Monitor antibody titer
Assess specificity
Determine optimal immunization schedule.
ELISA titration performed against the phospho-peptide and non-phospho peptide assesses antibody titer and specificity.
A terminal bleed is scheduled for bulk serum harvesting when:
Specific, high-affinity antibody titers reach a plateau (stabilize at a high level)
Antibodies bind at least 10 times more strongly to the phosphorylated peptide than to the non-phosphorylated peptide.
Affinity Purification and Negative Selection
Only a fraction of antibody species in the crude antiserum recognize the phospho‑epitope as the immunized animal generates a diverse mixture of antibodies. Column‑based affinity purification is performed to enrich desired phospho‑specific antibodies.
Positive Selection
It is a method that captures the antibodies that bind to the phospho-peptide. This is achieved by:
Immobilizing the phospho-peptide on a solid support
Passing the crude antiserum through the column and washing away unbound proteins
Eluting binding antibodies with a mild acidic buffer (pH 2.5–3.0) and immediately neutralizing them to preserve activity.
Negative Selection
Positive selection is followed by negative selection. This method removes unwanted antibodies that bind to non-phospho‑ peptides. This leaves only phospho-specific antibodies in the antiserum.
The purified antiserum is dialyzed into PBS or undergoes buffer exchange to:
Adjust pH and ionic strength
Remove unwanted chemicals
Ensure long-term storage stability
Prepare for downstream applications.
0.02% sodium azide is added for preservation. UV absorbance at 280 nm is used to determine the final concentration.
Rigorous Validation
It is important to ensure phospho‑specificity through rigorous validation that includes:
Peptide ELISA
ELISA is the primary tool that confirms whether the antibodies actually work as intended. This test plays a crucial role in quantifying binding strength (EC₅₀) and confirming phospho-specificity.
Quantifying Binding Strength (EC₅₀)
ELISA test determines how strongly antibodies bind to the phosphorylated peptide. Binding strength (EC₅₀) is calculated from serial dilutions of antibodies followed by binding signal measurements. A low EC₅₀ indicates high affinity.
Confirming Phospho-Specificity
Phospho-specificity confirmation requires two parallel ELISAs. One ELISA test is performed with the phospho-peptide coated on the plate and another with non-phospho peptide. Binding signal comparison of both tests confirms that the intended antibody binds strongly to the phosphorylated version but shows negligible binding to the non-phosphorylated version.
Western Blot ± Phosphatase
This validation test probes parallel cell or tissue lysates. The test involves two samples. One is treated with λ-phosphatase to remove phosphate groups, and another is left untreated.
The intended phospho-specific antibody loses signal in the phosphatase-treated sample and demonstrates strong signal in the untreated sample.
Signal loss may also occur due to protein degradation. Therefore, a pan-antibody control should show no change to confirm the signal loss is specifically due to dephosphorylation.
Cellular Modulation
The phosphorylation level of the target protein is changed to validate phospho-specific antibody function. The target phosphorylation state in cells is altered by applying specific kinase inhibitors or overexpressing a phosphatase.
The antibody loses or gains signal as phosphorylation levels change in the treatment. This reflects the antibody’s ability to detect biological changes within cells.
Immunofluorescence & Immunoprecipitation
These validation techniques prove that the antibody binds to its target and exhibits intended behaviour in biologically relevant systems.
Immunofluorescence (IF) ensures that the location detected by the antibody matches known biology. Co-staining with total-protein antibodies and using isotype controls checks for non-specific binding.
Immunoprecipitation (IP) validates an antibody’s ability to isolate a specific target protein and its direct interacting partners from cell lysates.
Conclusion
The rigorous validation of phospho-specific antibodies is paramount to ensure high affinity and specificity of the antibody. AAA Biotech offers a comprehensive range of extensively validated phospho-specific antibodies for researchers demanding antibodies with the highest standards of performance and reproducibility in their studies.
