Dataset Information

Specific, sensitive and quantitative protein detection by in-gel fluorescence.

ABSTRACT: Recombinant proteins in complex solutions are typically detected with tag-specific antibodies in Western blots. Here we describe an antibody-free alternative in which tagged proteins are detected directly in polyacrylamide gels. For this, the highly specific protein ligase Connectase is used to selectively fuse fluorophores to target proteins carrying a recognition sequence, the CnTag. Compared to Western blots, this procedure is faster, more sensitive, offers a better signal-to-noise ratio, requires no optimization for different samples, allows more reproducible and accurate quantifications, and uses freely available reagents. With these advantages, this method represents a promising alternative to the state of the art and may facilitate studies on recombinant proteins.

SUBMITTER: Fuchs ACD

PROVIDER: S-EPMC10154401 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Specific, sensitive and quantitative protein detection by in-gel fluorescence.

Fuchs Adrian C D ACD

Nature communications 20230502 1

Recombinant proteins in complex solutions are typically detected with tag-specific antibodies in Western blots. Here we describe an antibody-free alternative in which tagged proteins are detected directly in polyacrylamide gels. For this, the highly specific protein ligase Connectase is used to selectively fuse fluorophores to target proteins carrying a recognition sequence, the CnTag. Compared to Western blots, this procedure is faster, more sensitive, offers a better signal-to-noise ratio, req ...[more]

PMID: 37130834

Similar Datasets

Project description:BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

Dataset Information

Specific, sensitive and quantitative protein detection by in-gel fluorescence.

Publications

Specific, sensitive and quantitative protein detection by in-gel fluorescence.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets