|
|
Background: The current invention pertains to the development and utilization of an antibody that specifically recognizes tyrosine-phosphorylated PI3K p85 at position 452. The process encompasses antigen preparation, immunization, and the construction of affinity chromatography columns.
Methods: To facilitate efficient peptide conjugation to a carrier protein and subsequent peptide-based affinity purification, a cysteine residue (C) was incorporated at the C-terminus of the peptide as a linker. The final peptide sequence was identified as KLHEY(p)NTQFQE. The antibody was purified through a two-step affinity purification protocol: initially, the antiserum was passed through a phosphorylated peptide column to enrich phosphospecific antibodies, followed by passage through a non-phosphorylated peptide column to eliminate non-specific binders.
Results: This methodology enables the scalable production of the anti-phospho-PI3K p85 (Tyr452) antibody, which demonstrates high specificity for phosphorylation and strong affinity.
Conclusions: In comparison to traditional protein A purification, this approach is markedly more efficient.
DOI: 10.7754/Clin.Lab.2025.250804
|
