Conjugation kit

Manufactured by Thermo Fisher Scientific

The Conjugation kit is a laboratory product designed for the covalent attachment of molecules, such as proteins, peptides, or small molecules, to various carrier molecules or labels. The kit provides the necessary reagents and protocols to facilitate this conjugation process in a research or analytical setting.

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Lab products found in correlation

2 protocols using conjugation kit

Multicolor Flow Cytometric Analysis

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For detection and analysis, the following antibodies/clones were used: FcɛR1a (MAR-1; BioLegend); CD117 (2B8; BioLegend); CD45 (30-F11; BioLegend), β7 integrin (M293; BD Biosciences); CD11b (M1/70; BioLegend); CD11c (N418; BioLegend); CD4 (GK1.5; BioLegend); CD8 (53–6.7; BioLegend); CD103 (2E7; BioLegend); EPCAM (G8.8; BioLegend); Thy1.2 (53–2.1; BioLegend); CD31 (390; eBioscience); TGF-β1 (Tw7-16B4; BioLegend); and mMCP-1 (RF6.1; eBioscience). Anti-mMCP-1 and isotype control were conjugated in parallel to Alexa Fluor 647 using a conjugation kit (Life Technologies). Intracellular staining was conducted using a BD Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer-supplied protocol. For flow cytometry, the cells were collected and stained for surface markers for 45 min before fixation. Intracellular mMCP-1 staining was conducted overnight at 4°C. All cell sorting was on a BD FACSAria Fusion cell sorter using BD FACSDiva software. For all flow cytometry not involving cell sorting, data were collected on a BD LSRII Fortessa or BD CANTO-II using BD FACSDiva software. All downstream data analysis was conducted in FlowJo.

Derakhshan T., Samuchiwal S.K., Hallen N., Bankova L.G., Boyce J.A., Barrett N.A., Austen K.F, & Dwyer D.F. (2020). Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation. The Journal of Experimental Medicine, 218(1), e20200321.

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Vitronectin Conjugation and Characterization

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Human recombinant vitronectin was purchased from Advanced Biomatrix (Catalog #5052) and conjugated with Alexa Fluor 488 in-house using Alexa Fluor 488 carboxylic acid, succinimidyl ester (Life Technologies) at a 8:1 fluorophore to protein molar ratio in PBS (4 hrs, 4C). Following the reaction, the vitronectin solution was dialyzed against PBS for 48 hours with PBS changes every 12 hours to remove unreacted dye. The DOL for the vitronectin-Alexa Fluor 568 conjugate was calculated to be 6.25 using absorbance readings taken at 494 and 280 nm, as prescribed in the manufacturer’s manual provided with the conjugation kit from Life Technologies, and using 1.02.mL mg⁻¹ cm⁻¹ as the extinction coefficient for vitronectin^{63 (link)}. Three concentrations (40, 30 and 20 μg mL⁻¹) were tested to create a concentration vs. fluorescent intensity curve. Like with BSA, the stamps were plasma-treated before adsorption and the stamps were kept in contact with the dextran-coated wafers under weight for 20 mins.

Pushkarsky I., Tseng P., Warfe L., Black D., France B., Koziol-White C.J., Jester WF J.r., Trinh R.K., Lin J., Scumpia P.O., Morrison S.L., Panettieri RA J.r., Damoiseaux R, & Di Carlo D. (2018). Elastomeric sensor surfaces for high-throughput single-cell force biology. Nature biomedical engineering, 2(2), 124-137.

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