Custom Monoclonal Antibody Services

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A: (Cat. No. LA005) Monoclonal antibody services from gene to ascites Antibodies

1. Gene amplification, vector construction, protein expression in E. coli., immunization and sera collection, ELISA test
2. Splenocyte fusions are performed from mice with the best titers; The hybridoma cells are screened by ELISA
3. Standard 22-26-week antibody production schedule
4. Two of these 10 clones are selected for subcloning, isotype determination, ascites production and purification
5. Deliverables: construct vector, 1 mg protein, 50 ul pre-immune sera (1:10 dilution), 2 clones, 2-5 mg purified antibodies (2 clones), ELISA results >1:32,000, isotype determination

B: (Cat. No. LA006) Monoclonal antibody services from peptide design to ascites production

1. Include peptide design (free) and peptide synthesis, immunization and sera collection, ELISA test.
2. Splenocyte fusions and subclone, ascites production and purification.
3. Standard 20-26-week antibody production schedule
4. Deliverables: 2-3 mg peptides, 50 ul pre-immune sera, 2 clones, 2-5 mg purified antibodies (2 clones), ELISA results >1:32,000, isotype determination

C: (Cat. No. LA007) Monoclonal antibody services using phospho-peptide antigens

1. Peptide antigen design (free) and peptide synthesis
   • Phosphopeptide: >85% purity, 10 mg, 12-22 amino acids
   • Non-phosphopeptide: >85% purity, 10 mg, 12-22 amino acids
2. Conjugation of phosphopeptide to KLH or BSA (2-3 mg); conjugation of the non-phosphorylated peptide to BSA; immunization and sera collection; ELISA identification; fusion and hybridoma development; isotype determination; subcloning and cell cryopreservation; ascites production and purification (2 clones)
3. Standard 21-26 week antibody production schedule
4. Deliverables: 50 ul pre-immune sera, 2-3 mg peptides (unmodified and phospho), 2-5 mg phospho-specific antibodies (2 clones), ELISA results >1:32,000, isotype determination

D: Additional services:

• 4-week extension: We provide 1 additional immunization and 2 additional bleed collections for approximately 80-100ml of polyclonal antisera
• Affinity purification: Affinity purification of 25ml of sera including the affinity column for running additional purifications
• Antibody Conjugate Service: We can conjugate your antibody or protein to many labels: Biotin, Strepatvidin, HRP, AP, and fluorescent dyes.
• ELISA: On any bleed or purified antibody, upon request
• Western blot: Assay of antisera or purified antibodies against the antigens, upon request
• Immunochemistry: Assay of purified antibodies against the pathological slices, upon request

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Reference:

Jia Shen. et al. EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2. Nature 497, 383–387 (16 May 2013), doi:10.1038/nature12080

LifeTein helped designed and synthesized a series of phosphorylated peptides. Then the peptides were used for phospho-specific antibody productions. The phospo-specific antibodies by LifeTein were confirmed to react with the epidermal growth factor receptor (EGFR). The Hung's lab showed that AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells.

... The following peptides were chemically synthesized for antibody production in mice (Lifetein Conc.), Elisa verification (LifeteinConc.) and peptide competition assay in immunohistochemistry (IHC)... Supplementary information