The sequencing of antibody variable regions (VH and VL) from hybridomas or clonal B cells is a critical first step in numerous applications, including enabling production of recombinant antibody in various formats, antibody engineering, humanization, and affinity maturation. LakePharma can sequence variable heavy and light chain regions from hybridoma cell lines or clonal primary B cells from a variety of species. Following this service, the variable regions can be reformatted into whole IgGs or single-chain (scFv)-Fc and transiently expressed in mammalian cells, followed by confirmation of antigen binding.
Please contact LakePharma at firstname.lastname@example.org for custom quotes and details.
Cloning and sequencing antibody variable regions:
1. Extract and purify total RNA from hybridoma cells or clonal B cell.
2. Synthesize cDNA using optimized primer set.
3. Amplify the DNA fragments corresponding to the VL and VH using nested PCR.
4. Sequence the amplified DNA fragments. Amplified PCR fragments of the variable heavy and light chain sequences can be provided for an additional $100 plus shipping/handling.
For cloning and sequencing antibody V regions:
A. Greater than 100,000 cells in frozen vial(s) and shipped on dry ice
B. Isolated RNA samples dried on RNA isolation columns (before elution) and shipped on dry ice.
Note: please see FAQ: "For antibody variable region cloning, how should I prepare my hybridoma/B cell sample?"
Please include a hybridoma sample submission form.
1. Study report containing sequence analysis
2. Amplified PCR fragments of the variable heavy and light chain sequences can be provided for an additional $100 plus shipping/handling.