Abstract
Expression levels of cellular proteins can be affected by various perturbations, such as genetic knockout of interactors, drug treatments or cell stress. To specifically measure the effects on protein levels post-synthesis under different experimental conditions, it is important to compensate for transcriptional and other upstream changes. Here, we provide a protocol for a dual-fluorescence flowcytometry-based assay to determine protein levels. The protein of interest is genetically linked to enhanced GFP (eGFP) followed by a viral 2A self-cleaving peptide sequence and mCherry. As a result, translation of the reporter construct leads to two fluorescent protein products from the same mRNA template, which enables unambiguous protein expression analysis with normalization across samples.
Highlights
[Background] Synthesis and maintenance of cellular proteins depend on multiple processes, from transcriptional regulation, processing and degradation of mRNA to translation, folding, localization, posttranslational modification and protein degradation (Vogel and Marcotte, 2012)
The protein of interest is genetically fused to enhanced GFP followed by a viral 2A self-cleaving peptide sequence and a second fluorescent protein, mCherry (Figure 1)
The flow cytometry-based single-cell readout may enable the detection of more subtle differences in protein levels between experimental conditions, which could otherwise be masked in bulk readouts, such as immunoblot
Summary
1. 12-well cell culture plates (Fisher Scientific, catalog number: 12556005) 2. Falcon 5 ml Round-Bottom Tubes with 35 μm strainer lids (Corning, catalog number: 352235) 3. EMC4-KO 293FT cells (generated as described in PMID: 31516121) 5. Stbl[3] competent E. coli (MacroLab, catalog number: Stbl3) 6. EGFP coding sequence, amplified from pX458 (gift from Feng Zhang; Addgene plasmid, catalog number: 48138) 7. MCherry coding sequence, amplified from pCAGGS-mCherry (gift from Phil Sharp; Addgene plasmid, catalog number: 41583) 8. ADRB1 coding sequence, amplified from pcDNA3 Flag beta-1-adrenergic-receptor Robert Lefkowitz; Addgene plasmid, catalog number: 14698) 9. Dengue virus serotype 2 16681 strain NS4A-NS4B coding sequence, amplified from infectious clone (gift from Karla Kirkegaard, NCBI Reference Sequence: NC_001474) 10. Kapa HiFi HotStart ReadyMix (Kapa Biosystems, catalog number: KK2602) 11.
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