Antibody Sequencing
Sequencing of Variable Regions, Plus Leader Sequences of Both Heavy and Light Chains, From mAb Producing Cell Lines
Turnaround Time: 10 to 15 business days
Service Description:
• Extraction of total RNA from the cell pellet
• Reverse transcription and PCR amplification of heavy and light chains by 5′-RACE and 3′- RACE
• Specific murine and human constant domain primers are used to determine the isotype of the antibody
• Amplified products are purified and inserted into a sequencing vector
• Multiple independent colonies are sequenced for each chain to ensure accuracy (typically 8 colonies per chain)
Deliverables:
• Detailed project report with heavy chain variable region (VH) and light chain variable region (VL), plus leader sequences and alignments
• Plasmids with VH or VL, plus leader sequences
Suitable Sample Type:
•Frozen cells (1×107 cells) expressing target mAb
• EBV transformed B-cell line
• IgG expressing CHO or HEK 293 cell line
• Selected mAb producing hybridoma cell line
Monoclonal Antibody Sequencing
Poochon offers a rapid cDNA based de novo sequencing service for your valuable monoclonal antibodies. We can sequence from any antibody producing cell line with a service tailored to your requirements for quality control, patent applications, antibody engineering, and drug development. Our monoclonal antibody sequencing services are for sequencing of the variable domains of the heavy and light chain, the leader plus variable domains of the heavy and light chain, or the full length heavy and light chain from hybridomas and clonal B cells. Antibody types include IgG, IgM, IgA, and IgE antibody variable regions (VH and VL) from mouse, rat, hamster, rabbit, monkey, human, llama, alpaca, and camel. Plus, we offer protein amino acid sequence confirmation by LC-MS/MS.
