How to validate antibody VH and VL sequences in research?

Validation includes PCR amplification of VH/VL, colony PCR to confirm correct insertion, Sanger sequencing, and junction frame analysis to ensure productive rearrangements without frameshifts.

What do V/J/D gene usage and identity percentages indicate?

High identity to germline V/J (and D for heavy chain) genes together with a productive, in-frame junction indicates an intact, functional antibody variable region suitable for expression.

Why is the sequencing peak map important for antibody projects?

Clean, non-overlapping peaks confirm sequence accuracy and help rule out PCR/cloning-introduced errors, providing reliable templates for downstream expression and characterization.

Can AtaGenix assist with end-to-end antibody development?

Yes. AtaGenix supports design, cloning, recombinant expression, sequencing verification, and QC (e.g., WB/ELISA/SPR) to accelerate custom antibody discovery and validation.

Antibody Gene Sequencing and Validation Case Study | AtaGenix Custom Antibody Development

Release time: 2024-11-05 View volume: 682

Figure 1: Schematic of antibody coding gene structure, including 5’ and 3’ untranslated regions (UTRs). This illustrates the framework of heavy and light chain organization.

Figure 2: PCR Amplification of Heavy and Light Chain Variable Regions

Figure 2: PCR amplification of antibody heavy (VH) and light chain (VL) variable regions, showing clear amplification bands, confirming the successful cloning of variable regions for sequencing.

Figure 3: Colony PCR validation gel. M: DL10000 DNA Marker. The clear in-frame amplification bands confirm successful insertion of VH and VL sequences into the cloning vector.

Sequence	V-GENE and Allele	Functionality	V-REGION Identity (%)	J-GENE and Allele	D-GENE and Allele	AA Junction	Junction Frame
VH	Musmus IGHV1-71-16*01 F	Productive	96.53%	Musmus IGHJ4*01 F	Musmus IGHD2-4*01 F	CAITTGYAMDYW	In-frame
VL	Musmus IGKV4-74*01 F	Productive	97.51%	Musmus IGKJ4*01 F	-	CHQYYRSPFTF	In-frame

Figure 4: Antibody heavy and light chain sequence analysis. High sequence identity (>96%) confirms accurate cloning and alignment of VH and VL regions.

Figure 5: Antibody Heavy and Light Chain Sequencing Peak Map

Figure 5: Sequencing peak map of antibody VH and VL chains. Clear and consistent peaks demonstrate sequencing accuracy and reliable antibody variable region identification.

Need expert support for antibody cloning, sequencing, and validation? AtaGenix delivers end-to-end custom antibody services—from design to QC.

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Antibody Gene Sequencing and Validation Case Study | AtaGenix Custom Antibody Development

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