Western blots (WB) and peptide arrays (PA) were immunoblotted for various proteins. The following antibodies were used: GAPDH (1 : 5000 WB; Millipore, Burlington, MA, USA; MAB374), HA tag (1 : 500 IF, 1 : 1000 WB; Cell Signalling Technologies, Danvers, MA, USA; 2367), SUMO‐TnI (1 : 100 PA, WB, custom antibody produced in rabbits against SUMOylated synthetic peptide N‐HLKQVKKEDTEK‐C; Badrilla, Leeds, UK), SUMO1 (1 : 1000 PA, WB, Enzo BML‐PW9465‐0025), TnI (1 : 500 IF, 1 : 1000 WB; Abcam, Waltham, MA, USA; ab27003), Donkey anti‐mouse fluorescent secondary (1 : 5000 WB; Licor, Lincoln, NE, USA; 925–68 072), Donkey anti‐rabbit fluorescent secondary (1 : 5000 WB; Licor 925–68 073), Goat anti‐rabbit HRP secondary (1 : 5000 PA, WB; Sigma, Welwyn Garden City, UK; A6154), Alexa Fluor® 488 goat anti‐mouse (1 : 500 IF; Invitrogen, Waltham, MA, USA; A21071), and Alexa Fluor® 647 goat anti‐rabbit (1 : 500 IF; Invitrogen A21121).
Alexa fluor 647 goat
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 647 goat is a fluorescent labeling reagent used in various biomedical research applications. It is a conjugate of the Alexa Fluor® 647 dye and goat-derived antibodies. The Alexa Fluor® 647 dye is a far-red fluorescent dye with high brightness and photostability, making it suitable for sensitive detection applications.
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2 protocols using alexa fluor 647 goat
1
Immunoblotting for SUMOylated Proteins
2
Phagocytic Cell Visualization in Zebrafish Larvae
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Larvae were fixated on indicated time points overnight in 4% (v/v) paraformaldehyde (EMS, 100122) in PBS and stored in 100% methanol at −20 °C for immune‐histochemical staining and confocal imaging. Larvae were labelled with anti‐L‐plastin, which stains phagocytic cells according to established protocols (Bennett et al., 2001 ; Herbomel, Thisse, & Thisse, 1999 ). In short, larvae were rinsed with 1% PBTx, (1% Triton X‐100 in PBS), permeated in 0.24% trypsin in PBS and blocked for 3 hr in block buffer (10% normal goat serum [NGS] in 1% PBTx). Samples were incubated with anti‐L‐plastin (1:500 [v/v] dilution) in antibody buffer (PBTx containing 1% [v/v] NGS and 1% [w/v] BSA) overnight at RT. Samples were washed with PBTx, incubated for 1 hr in block buffer and stained with an Alexa‐Fluor‐647 goat‐anti‐rabbit as secondary antibody (Invitrogen A21070, 1:400), overnight at 4 °C.
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