Protein lysates in lysis buffer were mixed with 2× Laemmli buffer and heated to 95 °C for 5 min before being loaded onto a Precast Mini-Protean® TGX™ gels (456-9034, Biorad, Solna, Sweden). After separation, proteins were transferred to an Immobilon®-Fl membrane (IPFL0010, Millipore, Solna, Sweden). The membrane was blocked using the Intercept® (Tris Buffered Saline) blocking buffer (927-60001, Li-Cor, Bad Homburg, Germany) diluted 1:4 in PBS, and sequentially incubated with P2Y12 antibodies and goat-anti-rabbit Alexa 680 (A21088, ThermoFisher, Uppsala, Sweden) PBS + 0.1% Tween®20 or blocking buffer with 0.1% Tween®20 and 0.01% SDS, respectively. Beta-actin antibodies were used as loading control (ab6276, Abcam, Cambridge, UK) and incubated with IRDye 800CW Goat anti-mouse (Li-Cor, Bad Homburg, Germany,). All incubations were carried out at room temperature for 1 h or overnight at 4 °C. The membranes were scanned using an Odyssey scanner (Li-Cor, Bad Homburg, Germany).
Precast mini protean tgx gels
Manufactured by Bio-Rad
Sourced in Sweden, United States
Precast Mini-Protean® TGX™ gels are a type of lab equipment used for protein electrophoresis. These pre-cast polyacrylamide gels are designed for use in the Mini-Protean® electrophoresis system and provide a convenient solution for separating and analyzing protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon fl membrane, by Merck Group (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Ab6276, by Abcam (1 mentions)
Anti lamp1 antibody, by Cell Signaling Technology (1 mentions)
Goat anti rabbit hrp secondary antibody, by Jackson ImmunoResearch (1 mentions)
Tris glycine buffer, by Bio-Rad (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
2 protocols using precast mini protean tgx gels
1
Western Blotting of P2Y12 Receptor
2
SDS-PAGE and Western Blotting Protocol
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Precast Mini-PROTEAN® TGX™ gels, Immun-Blot® PVDF and supported nitrocellulose (NC) membranes (both of pore size 0.2 μm), Tris/glycine buffer, gelatin type-A from pig skin and all other routine chemicals required for SDS-PAGE and western blotting were purchased from Bio-Rad Laboratories (CA, USA). Halt™ protease inhibitor cocktail was purchased from Thermo Fisher Scientific (MA, USA). Bovine serum albumin (BSA) free of immunoglobulins and goat-anti-rabbit-HRP secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (PA, USA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (GA, USA). Nonfat dry milk powder (blotto) was purchased from a local grocery store. Anti-LAMP1 antibody was purchased from Cell Signaling Technology (MA, USA). Anti-Rab11a antibody was purchased from Santa Cruz Biotechnology (TX, USA), whereas the anti-cystic fibrosis transmembrane-conductance regulator (CFTR) antibody R3194 was a gift from CR Marino (VA Medical Center, TN, USA). General laboratory chemicals and reagents for tissue culture were purchased either from Sigma-Aldrich Chemical Co. (MO, USA) or Invitrogen (CA, USA).
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