Ez link maleimide activated horseradish peroxidase kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EZ-Link Maleimide Activated Horseradish Peroxidase Kit is a product designed for the covalent coupling of horseradish peroxidase (HRP) to sulfhydryl-containing molecules. The kit includes the necessary reagents and instructions to facilitate this conjugation process.

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Lab products found in correlation

Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Fm1 43, by Thermo Fisher Scientific (1 mentions) Phrodo dextran, by Thermo Fisher Scientific (1 mentions) Atto 647n maleimide, by ATTO-TEC (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Pd minitrap g 25 column, by GE Healthcare (1 mentions) Pd mini trap, by Cytiva (1 mentions) Microplate reader, by Agilent Technologies (1 mentions)

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Horseradish peroxidase (215 citations) Horseradish peroxidase kit (3 citations)

3 protocols using ez link maleimide activated horseradish peroxidase kit

Fluorescent Protein Labeling Protocols

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The rabbit anti-synaptobrevin-2 (VAMP2) antibody was obtained from Synaptic Systems (#104 202). Alexa Fluor secondary antibodies, together with maleimide dyes, pHrodo-dextran, Alexa Fluor labeled CTB and FM1-43 were purchased from Invitrogen. Atto647N maleimide was obtained from Atto Tec. The remaining reagents were obtained from Electron Microscopy Sciences or Sigma Aldrich unless otherwise specified. The expression, purification and fluorescent labeling of BoNT/A-Hc was carried out as previously described1 (link)37 (link). BoNT/A-Hc was conjugated to HRP using the EZ-Link Maleimide Activated Horseradish Peroxidase Kit (Thermo Scientific), while BoNT/A-Hc and CTB were conjugated to colloidal gold as described previously1 (link). The anti-GFP nanobody (kind gift from Kirill Alexandrov) was conjugated to pHrodo-NHS-ester.

Harper C.B., Papadopulos A., Martin S., Matthews D.R., Morgan G.P., Nguyen T.H., Wang T., Nair D., Choquet D, & Meunier F.A. (2016). Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles. Scientific Reports, 6, 19654.

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BoNT/A-Hc Protein Conjugation Protocols

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His6-tagged BoNT/A-Hc was purified using Co IDA-agarose (Scientifix) as described previously (Couesnon et al., 2009 (link); Harper et al., 2011 (link)), then conjugated to Atto647N maleimide (Atto-TEC, catalog #647N-41) according to the manufacturer’s instructions. For BoNT/A-Hc-HRP conjugation, the EZ-Link Maleimide Activated Horseradish Peroxidase Kit (Thermo Scientific, catalog #31494) was used to conjugate HRP to sulfhydryl groups of BoNT/A-Hc. A 1:2 molar ratio of BoNT/A-Hc and activated HRP were incubated in conjugation buffer at 4°C overnight. The conjugated BoNT/A-Hc-HRP was purified using a PD MiniTrap G-25 Column (GE Healthcare, catalog #28-9180-08) following the gravity protocol. The protein concentration was measured using the Bradford reagent (Bio-Rad, catalog #500-0006). All conjugated proteins were stored at −80°C with the addition of 0.1% bovine serum albumin (BSA) as a stabilizer.

Wang T., Martin S., Papadopulos A., Harper C.B., Mavlyutov T.A., Niranjan D., Glass N.R., Cooper-White J.J., Sibarita J.B., Choquet D., Davletov B, & Meunier F.A. (2015). Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A. The Journal of Neuroscience, 35(15), 6179-6194.

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PEDV Virion Binding Quantification

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Ninety six-well plates (Griener, Frickenhausen, Germany) were firstly coated by PEDV at a density of 10⁶ virions/well at 4 °C overnight, followed by a 1 h block with 4% BSA. After washed with PBS for three times and added with 100 μL diluted mAb (1:100), the sample was incubated for 1 h at 37 °C. Next, the plates were rinsed with PBS and cultured for 1 h with mAb 2G8 which labelled with horseradish peroxidase by EZ-Link Maleimide Activated Horseradish Peroxidase Kit (Thermo scientific, USA). The reacting results were visualized using tramethylbenzidine (TMB), stopped by HCL. All samples were repeated three times. The absorbance was determined using a microplate reader (Bio-Tek) at 450 nm.

Gong L., Lin Y., Qin J., Li Q., Xue C, & Cao Y. (2018). Neutralizing antibodies against porcine epidemic diarrhea virus block virus attachment and internalization. Virology Journal, 15, 133.

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