Seiler, Kristina; Rafiq, Sreoshee; Tschan, Mario P (2023). Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging. Bio-protocol, 13(14), e4777. bio-protocol 10.21769/BioProtoc.4777
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Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader. Graphical overview.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
04 Faculty of Medicine > Service Sector > Institute of Pathology > Tumour Pathology 04 Faculty of Medicine > Service Sector > Institute of Pathology |
Graduate School: |
Graduate School for Cellular and Biomedical Sciences (GCB) |
UniBE Contributor: |
Seiler, Kristina, Rafiq, Sreoshee, Tschan, Mario Paul |
Subjects: |
500 Science > 570 Life sciences; biology 600 Technology > 610 Medicine & health |
ISSN: |
2331-8325 |
Publisher: |
bio-protocol |
Language: |
English |
Submitter: |
Pubmed Import |
Date Deposited: |
27 Jul 2023 16:04 |
Last Modified: |
20 Dec 2023 12:11 |
Publisher DOI: |
10.21769/BioProtoc.4777 |
PubMed ID: |
37497448 |
Uncontrolled Keywords: |
Antibody-free detection Endogenous tagging HiBiT Protein expression Protein isoform Split-luciferase |
BORIS DOI: |
10.48350/185087 |
URI: |
https://boris.unibe.ch/id/eprint/185087 |
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