Post-translational modifications (PTMs) are the master switches of cell signaling, regulating everything from protein stability and localization to interaction networks and enzymatic activity. However, the transient and subtle nature of these chemical groups makes them exceptionally difficult targets for conventional antibody generation. Creative Biolabs leverages our advanced Phage Display for Challenging Target Discovery to overcome these hurdles. By utilizing precisely designed neo-epitope antigens and rigorous subtractive biopanning strategies, we isolate high-affinity antibodies capable of distinguishing a single phosphate, methyl, or ubiquitin group with exquisite specificity.
Proteins are rarely static entities; their functions are dynamically modulated by a vast array of PTMs, including phosphorylation, methylation, acetylation, ubiquitination, and glycosylation. These modifications create "neo-epitopes"—unique structural features that appear only when the protein is modified. Detecting these modification-state dependent binders is crucial for deciphering complex biological pathways, such as the histone code in epigenetics or kinase cascades in cancer signaling.
Despite their importance, generating PTM-specific antibodies is fraught with challenges:
Phage display technology bypasses the limitations of the immune system, allowing for the in vitro selection of binders from billions of variants against synthetic peptides carrying the specific modification, ensuring the discovery of highly selective probes.
We provide a comprehensive suite of services tailored to target various classes of post-translational modifications.
Phosphorylation is the most common PTM in signal transduction. We generate antibodies that specifically recognize phosphorylated serine (pSer), threonine (pThr), or tyrosine (pTyr) residues within a specific sequence context, enabling the precise monitoring of kinase activity.
Crucial for epigenetics research, these antibodies target specific histone modifications (e.g., H3K4me3, H3K27ac). Our screening platform can distinguish between mono-, di-, and tri-methylation states, providing high-resolution tools for chromatin analysis.
Targeting the ubiquitin-proteasome system requires antibodies that recognize specific poly-ubiquitin linkage types (e.g., K48 vs. K63) or the junction between the modifier protein and the substrate, facilitating the study of protein degradation and stability.
Beyond natural PTMs, we apply our platform to generate site specific antibodies against point mutations (e.g., oncogenic drivers like KRAS G12C) or drug-induced adducts, supporting precision oncology and toxicology studies.
Our PTM antibody generation pipeline utilizes rigorous subtractive selection to ensure the final binders recognize only the modified state.
We synthesize short peptides (10-20 amino acids) containing the specific PTM of interest (Positive Antigen). Crucially, we also synthesize the exact same sequence without the modification (Negative/Control Antigen). Both are conjugated to carrier proteins (e.g., BSA, KLH) or biotinylated for surface immobilization.
To ensure high specificity, we use a 'subtractive biopanning' strategy. First, we filter our phage library (scFv, Fab, or VHH) against the unmodified peptide to remove any antibodies that bind to the backbone. We then screen the remaining pool against the modified target. This ensures that the final antibodies recognize only the specific modification.
Individual clones are screened by dual-ELISA. We compare the signal against the modified peptide versus the unmodified control. Only clones showing high affinity for the PTM and negligible binding to the control are selected for sequencing and further analysis.
Top candidates are produced as soluble antibodies and validated in relevant biological assays, such as Western Blotting using lysates from treated vs. untreated cells, or Immunofluorescence to verify subcellular localization.
Mapping the "histone code" by identifying specific methylation, acetylation, and phosphorylation combinations on histone tails that regulate gene expression.
Monitoring the activation state of kinase pathways (e.g., MAPK, PI3K/Akt) by detecting specific phosphorylation events on downstream effectors.
Developing diagnostic tools that detect aberrant PTMs associated with disease states, such as hyperphosphorylated Tau in Alzheimer's disease.
The specificity achievable with phage display for PTMs is well-documented. In a study focused on generating binders against a specific phosphohistidine modification (a notoriously unstable PTM), researchers utilized a humanized antibody library. Through rigorous subtractive panning against non-phosphorylated analogs, they isolated a clone (hSC44.20N32FL) that demonstrated a ~10-fold higher affinity for the 3-phosphohistidine target compared to the parental antibody. Crucially, the ELISA data confirmed that the selected antibody bound strongly to the phosphorylated histidine peptide while showing negligible binding to the non-phosphorylated control or other phosphate isomers, validating the power of phage display to discriminate subtle chemical modifications.
Fig.1
Phage display selection and specificity validation of phosphohistidine antibodies.1
Q: How do you ensure the antibody binds the PTM and not the peptide sequence?
Q: Can you generate antibodies against multiple PTMs on the same protein?
A: Yes, we can synthesize peptides containing multiple modifications (e.g., dual phosphorylation) to generate antibodies that recognize specific combinatorial states.
Q: What is the success rate for difficult PTMs like methylation?
A: While methylation is challenging due to its small size, our optimized libraries and high-sensitivity screening protocols have successfully yielded high-affinity methyl-specific binders for histone marks.
"Generating an antibody specific to a single phosphorylation site on our kinase target seemed impossible due to high sequence homology. The subtractive panning strategy Creative Biolabs proposed was exactly what we needed. The final antibody has high affinity for the p-site with zero cross-reactivity to the non-phosphorylated control. Excellent work."
"Our epigenetics project required a binder for a specific histone methylation mark. Since the target is highly conserved, traditional animal immunization failed us repeatedly. Their phage display platform overcame the immune tolerance issue and we received a highly specific binder that works beautifully in our chromatin analysis assays."
"We approached them for a K48-linkage specific ubiquitin antibody. The team's expertise in antigen design was impressive—they synthesized the complex neo-epitope perfectly. We got valid clones in about 6 weeks. It is rare to find a CRO that understands ubiquitin signaling mechanics this well."
"Differentiation between a point mutation and the wild-type protein is tricky. Creative Biolabs managed to isolate a conformational antibody that strictly recognizes the mutant neo-epitope. The data provided in the report was very convincing and saved us a lot of internal validation time. Highly recommended."
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