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De novo Antibody Sequencing Service
$1200 for PCR-based cloning and sequencing of mouse hybridoma IgG antibodies if hybridoma or mRNA/cDNA from hybridoma is available. Prices are higher for cloning and sequencing antibodies from rat, hamster, rabbit, moneky, and human hybridomas or clonal B cells.
De novo protein sequencing of monoclonal antibodies (mAbs) is required when neither the original cell line nor mRNA/cDNA is available, or when characterization of post-translational modifications is needed to verify antibody integrity and effectiveness. Different from sequencing confirmation by peptide mapping where the protein sequence is already known and is used as a guideline to confirm the amino acid sequence, de novo antibody sequencing can be used to discover the amino acid sequence of an antibody without prior knowledge of any DNA or protein sequence. De novo antibody sequencing involves deliberately fragmenting intact peptides within a mass spectrometer in order to get structural information from fragmentation.
If only proteins are available, the intact amino acid sequence of variable and/or constant regions of monoclonal antibodies can be otained by our do novo antibody sequencing service.
General procedure of de novo antibody sequencing service:
A. Preliminary digestion with trypsin followed by LC-MS/MS;
B. Scale-up digestion with trypsin, Asp-N, Glu-C, and/or combination of all three enzymes followed by fragmentation optimization;
C. If required, separation of antibody heavy chain (HC) and light chain (LC) before multiple enzymatic digestions described in B followed by LC-MS/MS;
D. Data analysis.
Sample requirements for de novo antibody sequencing service:
Prerequisites- services required to determine feasibility of de novo antibody sequencing:
A. Intact Mass Analysis by LC-MS;
B. RP-HPLC Analysis;
Sample size: 3-5 mg at 1 mg/ml preferred (minimum: 2 mg at 1 mg/ml);
Formulation: freeze-dried or dissolved in 1X PBS or equivalent buffers;
Purity: > 95%;
Filtration: filtered with 0.22 um filters.
Information required from clients:
A. Source of antibody (e.g., mouse ascites or commercial mAb);
B. Isotype (if known);
C. Formulation (buffer type, pH, molar concentration, any additives, and protein concentration);
D. Any information on solubility and/or stability of the test article.
A. Primary amino acid sequences of variable regions (VH and VL) of antibody heavy and light chains;
B. Purified 3 mg scale transient production of recombinant monoclonal antibodies;
C. Study report.
We also provide the following antibody sequencing services:
Cloning and Sequencing Heavy- and Light-chain Variable Regions (VH and VL) of Hybridoma or Clonal B Lines
Single B Cell Antibody Cloning and Sequencing Service
N-terminal Protein Sequencing Service
Sequence Liability Identification Service