Coomassie staining

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Coomassie staining is a laboratory technique used to detect and visualize proteins in gel-based electrophoresis experiments. It employs a dye called Coomassie brilliant blue that binds to proteins, allowing their identification and quantification.

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12 protocols using coomassie staining

Quantification of Protein Expression by SDS-PAGE

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In order to quantify YFP produced, cultures at stationary growth step were harvested at 4,000 × g for 15 min at 4°C. Cells were disrupted by sonication for 2 min (40 s each pulse) using a Vibra Cell sonicator (Sonicator Sonics & Materials, Newton, United Kingdom). Lysates were analyzed by electrophoresis SDS-PAGE with 10% acrylamide gels using Mini-PROTEAN Tetra Cell (Biorad, California, CA, United States) followed by Coomassie staining (Fisher Scientific, Madrid, Spain). Standard curve was constructed to calculate YFP concentration by densitometric analysis using ImageJ Gel Analyzer software (Rueden et al., 2017 (link)).

Lozano Terol G., Gallego-Jara J., Sola Martínez R.A., Martínez Vivancos A., Cánovas Díaz M, & de Diego Puente T. (2021). Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21. Frontiers in Microbiology, 12, 682001.

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Protein Quantification and Visualization

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Protein concentrations were measured using the micro BCA protein assay (ThermoFisher, IL, USA). Whole-cell and mitochondrial protein extracts (15 µg) were resolved on conventional 10% SDS-PAGE gels and transferred to PROTRAN^®® nitrocellulose membranes (Whatman GmbH, GE Healthcare Life Sciences, Germany). Secreted protein extracts (25 µg) were separated either on conventional 10% SDS-PAGE gels and visualized by Coomassie staining (Fisher Bioreagents, Geel, Belgium), or, alternatively, they were resolved on 10% Mini-Protein TGX Stain Free Precast Gels (Bio-Rad, Mory, France) followed by quantification of the fluorescent protein signals in a ChemiDoc™ MP Imager (Bio-Rad, CA, USA).

García-Bartolomé A., Peñas A., Illescas M., Bermejo V., López-Calcerrada S., Pérez-Pérez R., Marín-Buera L., Domínguez-González C., Arenas J., Martín M.A, & Ugalde C. (2020). Altered Expression Ratio of Actin-Binding Gelsolin Isoforms Is a Novel Hallmark of Mitochondrial OXPHOS Dysfunction. Cells, 9(9), 1922.

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Evaluating YFP Protein Solubility

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To evaluate the solubility of the overexpressed YFP protein, chemically competent cells harboring constructed pSF-pMB1′-YFP or pSF-p15A-YFP vectors were grown overnight in batch mode at 37°C with orbital shaking (250 rpm). Culture medium and inductor concentration were selected in expression analysis. Pellets were harvested by centrifugation (20 min; 4,500 × g) and resuspended in native buffer (50 mM K₂HPO₄, 500 mM NaCl, pH 8). Cells were disrupted by sonication for 2 min (40 s each pulse) using a Vibra Cell sonicator (Sonicator Sonics & Materials, Newton, United Kingdom). The lysates were clarified by centrifugation at 14,000 × g for 30 min at 4°C to obtain supernatants (soluble protein extracts). Pellets were resuspended again with denatured buffer (50 mM K₂HPO₄, 500 mM NaCl, urea 6 M, pH 8) and incubated under shaking for 30 min. Finally, cells were centrifugated at 14,000 × g for 30 min at 4°C to isolate supernatants (insoluble protein extracts). To analyze YFP solubility, electrophoresis SDS-PAGE with 10% acrylamide gels was carried out. Protein gels were run under denaturing conditions using Mini-PROTEAN Tetra Cell (Biorad, California, CA, United States) followed by Coomassie staining (Fisher Scientific, Madrid, Spain). ImageJ Gel Analyzer software was used for densitometric quantification (Rueden et al., 2017 (link)).

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Chikungunya Virus Interaction Proteomics

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293T cells were transfected with pCMV6/myc-DDK-SK2 or pCMV6 (empty vector) using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). At 24-h post-transfection the cells were either mock infected or infected with CHIKV at a MOI of 5 for 24 h. As a control pCMV6/myc-DDK-SK2 transfected cells were mock infected, and as an additional control, cells were transfected with an empty vector and also infected. After 24 h, the cell lysates were prepared in lysis buffer (same as above). Myc-DDK-SK2 complexes were precipitated using a myc monoclonal antibody cross-linked to agarose beads (Sigma, St. Louis, MO, USA). The precipitated material was analyzed by SDS-PAGE analysis followed by Coomassie staining (Thermo Fisher Scientific, Middletown, VA, USA) and prepared for LC-MS/MS analysis. Instrumentation consisted of an Ultimate 3000 Nano-LC connected to a Thermo Scientific LTQ Orbitrap Elite using a data-dependent method consisting of a 30 000 resolution MS1 scan followed by 15 ms/ms rapid scans of the highest intensity ions. Searches were performed with MASCOT 2.4 using the SwissProt_2014_01 database with human taxonomy specified. The false discovery rate was set at 0.5%. Mass tolerances were 10 ppm for the MS1 scan and 200 ppm for all ms/ms scans.

Reid S.P., Tritsch S.R., Kota K., Chiang C.Y., Dong L., Kenny T., Brueggemann E.E., Ward M.D., Cazares L.H, & Bavari S. (2015). Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex. Emerging Microbes & Infections, 4(10), e61-.

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Protein Characterization by Spectroscopy

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All protein concentrations were determined by the Bradford assay, and protein purity was assessed by SDS-PAGE with Coomassie staining (Thermo Fisher Scientific). Metal analysis of the as-purified proteins was determined by inductively coupled atomic emission spectrometry (ICP-AES). Fe content was determined colorimetrically as described (58 ).

Ueda C., Langton M., Chen J, & Pandelia M.E. (2022). The HBx protein from hepatitis B virus coordinates a redox-active Fe-S cluster. The Journal of Biological Chemistry, 298(4), 101698.

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Peptide Degradation by DPP Enzymes

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Lyophilized peptide YY(1–36), peptide YY(3–36), neuropeptide Y, and Human Chemokine CXCL12 were resuspended in water to 100 μM and stored at −20°C. Enzymatic activity was confirmed by GP-AMC assay. Reactions were prepared with 8 μL of 100-μM peptide stock, 8 mL of 100 nM enzyme (DPP4, DPP8 and DPP9) or PBS (for control), and 64 μL of PBS (Corning). Reactions incubated at 37°C for 24 h. 9 μL aliquots were taken at 0, 1, 4, 8 and 24 h and immediately boiled to deactivate the enzyme. 1 μL of 100 mM 1 was added to the aliquots. After 16 h of incubating at 37°C, an equal volume of 2× loading dye was added, samples were boiled and run on 16% tricine gels (Invitrogen) at 125 V for 90 min. A semi-dry transfer at 25 V, 2.5 A for 15 min was used to transfer proteins to the membrane, which was then blocked and blotted for biotin with streptavidin IR dye. Additional 9 03bcL aliquots were also taken at 0, 24 h for Coomassie staining (Invitrogen) according to the manufacturer’s specifications.

Griswold A.R., Cifani P., Rao S.D., Axelrod A.J., Miele M.M., Hendrickson R.C., Kentsis A, & Bachovchin D.A. (2019). A chemical strategy for protease substrate profiling. Cell chemical biology, 26(6), 901-907.e6.

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Protein Digestion and Separation

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Prior to protein digestion, equal amounts of protein (80 µg) from unlabelled and labelled samples were combined. Samples were reduced in 10 mM DTT and alkylated in 50 mM Iodoacetamide prior to boiling in loading buffer, and then separated by one-dimensional SDS-PAGE (4–12 % Bis–Tris Novex mini-gel, Invitrogen) and visualised by colloidal Coomassie staining (Novex, Invitrogen). The entire protein gel lanes were excised and cut into 10 slices each. Every gel slice was subjected to in-gel digestion with trypsin overnight at 37 °C. The resulting tryptic peptides were extracted by formic acid (1 %) and acetonitrile (CH₃CN), lyophilized in a speedvac and resuspended in 1 % formic acid.

Zhang H., Angelopoulos N., Xu Y., Grothey A., Nunes J., Stebbing J, & Giamas G. (2015). Proteomic profile of KSR1-regulated signalling in response to genotoxic agents in breast cancer. Breast Cancer Research and Treatment, 151(3), 555-568.

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Di-scFv Purification and Characterization

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For phospho-53BP1 foci formation and trypan blue exclusion assays, di-scFv variant samples were exchanged into respective growth media (i.e. DMEM or RPMI 1640) and sample concentration was quantified using the Bradford assay. Subsequently, SDS-PAGE with Coomassie staining (Invitrogen) was performed to confirm di-scFv stability in the samples. All samples were filter sterilized using a 0.2 µm pore-sized syringe filter, and the concentration of di-scFv in PBS was quantified using absorbance at 280 nm with a Nanodrop 1000 (Thermo Fisher Scientific).

Rattray Z., Dubljevic V., Rattray N.J.W., Greenwood D.L., Johnson C.H., Campbell J.A, & Hansen J.E. (2018). Re-engineering and evaluation of anti-DNA autoantibody 3E10 for therapeutic applications. Biochemical and biophysical research communications, 496(3).

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Quantifying AAV-Delivered Antibodies in SIV Infection

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To measure the concentration of 5L7 IgG1 and 4L6 IgG1 in vivo we performed a SIVmac239 gp140 (Immune Tech)/anti-rhesus IgG ELISA (Southern Biotech). Absorbance at 450 nm was compared to a serial dilution of purified mAb produced in HEK 293T cells, and the amount of antibody in serum was extrapolated based on the mAb standard curve. Reference protein was quantified by NanoDrop (Thermo Scientific) A₂₈₀ measurement and purity was verified by Coomassie staining (Life Technologies). Levels of AAV-delivered antibody were measured up to the time of SIV challenge: 44 weeks (5L7 IgG1) and 14 weeks (4L6 IgG1) after AAV administration. The appearance of anti-env antibody responses following SIV infection obviated our ability to measure levels of delivered mAb at post-infection time points.

Fuchs S.P., Martinez-Navio J.M., Piatak M J.r., Lifson J.D., Gao G, & Desrosiers R.C. (2015). AAV-Delivered Antibody Mediates Significant Protective Effects against SIVmac239 Challenge in the Absence of Neutralizing Activity. PLoS Pathogens, 11(8), e1005090.

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Purification of rAAV Proteins from HEK293T

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HEK293T cells (ATCC) were transfected with rAAV vector plasmids and proteins were purified from cell culture supernatant using Protein A Plus (Pierce). Concentration of purified proteins was determined by NanoDrop (Thermo Scientific) A₂₈₀ measurement and purity and integrity was verified by Coomassie staining (Life Technologies).

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