Why Use Tag Knockin Models
Overcoming Antibody Limitations
High quality antibodies are not available for every protein of interest. Even when antibodies exist, they may not work in all applications such as immunoprecipitation, ChIP, or live cell imaging. Tag knockin models provide standardized detection reagents that work reliably across multiple applications.
Physiological Expression Levels
Unlike overexpression approaches, tag knockin preserves endogenous regulatory control. The tagged protein is expressed at normal levels from the native promoter, avoiding artifacts associated with ectopic or overexpressed constructs.
Consistent Detection Across Conditions
Epitope tag antibodies work identically regardless of the tagged protein's identity. This enables standardized protocols for detection and purification that work reliably across different target proteins.
Common Epitope Tags
FLAG Tag
DYKDDDDKThe FLAG tag is an 8 amino acid peptide with excellent antibody availability and low immunogenicity. FLAG tagged proteins can be detected by immunoblotting, immunofluorescence, and flow cytometry, and purified by anti FLAG affinity chromatography.
Multiple FLAG copies (3xFLAG) increase detection sensitivity for low abundance proteins.
Learn moreHA Tag
YPYDVPDYAThe HA tag derives from influenza hemagglutinin and provides robust detection with widely available antibodies. HA is particularly useful for immunofluorescence and immunoprecipitation applications.
Learn moreV5 Tag
GKPIPNPLLGLDSTThe V5 tag from simian virus 5 provides sensitive detection with minimal cross reactivity. V5 is often used when multiple tagged proteins need to be distinguished in the same experiment.
Myc Tag
EQKLISEEDLThe Myc tag from c-Myc provides excellent immunoprecipitation performance and works well for protein interaction studies.
His Tag
HHHHHH (6xHis)Polyhistidine tags enable affinity purification using nickel or cobalt resins. His tags are particularly useful when large scale protein purification from tissue is required.
Tag Placement Considerations
N Terminal Tagging
N terminal tags are positioned immediately after the start codon. This placement is appropriate when the protein's C terminus is functionally important or when the N terminus is accessible for antibody binding.
N terminal tagging may interfere with signal peptide cleavage for secreted proteins or with N terminal modifications.
C Terminal Tagging
C terminal tags are positioned immediately before the stop codon. This placement is appropriate when the protein's N terminus is functionally important or when the C terminus is accessible.
C terminal tagging may interfere with C terminal processing or with GPI anchor addition for membrane proteins.
Internal Tagging
For proteins where both termini are functionally important, internal tag placement in a flexible loop region may preserve protein function while enabling detection.
Linker Sequences
Flexible linker sequences between the tag and the protein can improve accessibility for antibody binding and reduce steric interference with protein function.
Applications of Tag Knockin Models
Protein Detection and Localization
Tag knockin enables detection of endogenous proteins in tissues and cells using anti-tag antibodies. This is particularly valuable when gene-specific antibodies are unavailable or unreliable.
Protein Purification
Affinity purification using anti-tag resins enables isolation of endogenous protein complexes for mass spectrometry analysis, enzymatic studies, or structural biology.
Chromatin Immunoprecipitation
ChIP with anti-tag antibodies enables mapping of transcription factor binding sites when ChIP grade antibodies for the protein of interest are unavailable.
Protein Interaction Studies
Immunoprecipitation of tagged proteins followed by mass spectrometry identifies interacting partners under physiological conditions.
Live Imaging
Fluorescent protein tags enable live imaging of protein dynamics. GFP, mCherry, tdTomato, and other fluorescent tags can be knocked in to enable real time visualization.
Learn moreTag Knockin Design Considerations
Allele Architecture
Tag knockin alleles are designed to insert the epitope tag sequence in frame with the target protein coding sequence.
Validation Strategy
Tagged protein expression is validated by immunoblotting and immunofluorescence using anti-tag antibodies. Comparison with untagged littermates confirms specificity of detection.
Selected Publications
Tag knockin models from ingenious targeting laboratory have enabled protein studies published in leading journals:
Tebbe L, Mwoyosvi ML, Crane R, Makia MS, Kakakhel M, Cosgrove D, Al-Ubaidi MR, Naash MI. (2023).
The usherin mutation c.2299delG leads to its mislocalization and disrupts interactions with whirlin and VLGR1. ↗Nat Commun 14(1): 972
Rumney RMH, Róg J, Chira N, Kao AP, Al-Khalidi R, Górecki DC. (2022).
P2X7 Purinoceptor Affects Ectopic Calcification of Dystrophic Muscles. ↗Front Pharmacol 13: 935804
Samant SA, Pillai VB, Gupta MP. (2021).
Skeletal muscle-specific over-expression of the nuclear sirtuin SIRT6 blocks cancer-associated cachexia by regulating multiple targets. ↗JCSM Rapid Commun 4(1): 40-56
What Researchers Say
“The people at InGenious are friendly, professional, and extremely good at what they do. I have made 5 Knockin mice with them and everything has gone like clockwork.”
— David B. Roth, MD, PhD
Perelman School of Medicine, University of Pennsylvania
Start Your Project
Ready to discuss a tag knockin model for your protein of interest? Our scientific team provides complimentary consultation to help you design the optimal targeting strategy.
Frequently Asked Questions
Tag Knockin Technology Insights
Learn about epitope tagging strategies, protein detection methods, and practical applications for tag knockin models. Expert guidance from our PhD scientists.