Gel electrophoresis of FACS-purified cell protein lysates was run on 4–12% NuPAGE Bis-Tris gradient gel (Thermo Fisher) and transferred to nitrocellulose membrane (Amersham). Membrane was blocked in TBS containing 2% bovine serum albumin (BSA) and 0.1% Tween-20 for 1 hr at RT, incubated with primary antibodies overnight at 4°C and with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 hr at RT. HRP activity was detected using enhanced chemiluminescence (ECL) substrate (Amersham). Quantification of bands was performed on ImageJ software. The following antibodies were used: ECAD (rabbit, 1:1000, Cell Signaling, AB_2291471), PCAD (goat, 1:1000, R and D Systems, AB_355581), p120 (mouse, 1:1000, Zymed, AB_87165), β-catenin (mouse, 1:1000, BD Biosciences, AB_11127855), α-catenin (rabbit, 1:4000, Sigma, AB_476881), claudin 1 (rabbit, 1:500, Abcam, AB_301644), ZO1 (rabbit, 1:500, Life Technologies, AB_2533938) and GAPDH (mouse, 1:4000, Abcam. AB_2107448).
Enhanced chemiluminescence ecl substrate
Manufactured by Cytiva
Sourced in United Kingdom
Enhanced chemiluminescence (ECL) substrate is a laboratory reagent used in Western blotting techniques to detect and quantify target proteins. It catalyzes a light-emitting chemical reaction that can be captured and measured, enabling the visualization and analysis of protein levels in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Horm collagen, by Takeda Pharmaceuticals (1 mentions)
U46619, by Cayman Chemical (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
5 n ethylcarboxamidoadenosine neca, by Bio-Techne (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cytiva (1 mentions)
Oregon green 488 bapta 1 am, by Thermo Fisher Scientific (1 mentions)
Syk p525 526, by Cell Signaling Technology (1 mentions)
Chronolume, by Chrono-Log (1 mentions)
2 protocols using enhanced chemiluminescence ecl substrate
1
Western Blot Analysis of Cell-Cell Adhesion Proteins
2
Platelet activation signaling pathway
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Horm collagen was obtained from Takeda (High Wycombe, UK). NECA (5'-Nethylcarboxamidoadenosine) was purchased from Tocris Bioscience (Bristol, UK). Cangrelor was purchased from Medicines Company. U46619 was obtained from Cayman Chemical.
Chronolume and ATP were from Chrono-log Corporation (Manchester, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies and Enhanced Chemiluminescence (ECL) substrate were obtained from Amersham Biosciences (GE Healthcare, Bucks, UK). Oregon green 488 BAPTA-1-AM was purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). Other reagents were obtained from Sigma.
Antibodies: PLCγ2 p1217, PLCγ2 p759, Syk p525/526, Syk p323, Syk p352, LAT p132 and LAT p171 were from Cell Signalling Technology (Danvers, Massachusetts, United States).
LAT p200 mAb was obtained from Abcam (Cambridge, UK). Mouse anti-human antiphosphotyrosine (clone 4G10) mAb was from Millipore UK Ltd (Watford, UK). Alexa Fluor 488 phalloidin and Alexa Fluor 647 were purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). 1G5-Fab recognises monomeric and dimeric GPVI was raised as described 23 .
Chronolume and ATP were from Chrono-log Corporation (Manchester, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies and Enhanced Chemiluminescence (ECL) substrate were obtained from Amersham Biosciences (GE Healthcare, Bucks, UK). Oregon green 488 BAPTA-1-AM was purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). Other reagents were obtained from Sigma.
Antibodies: PLCγ2 p1217, PLCγ2 p759, Syk p525/526, Syk p323, Syk p352, LAT p132 and LAT p171 were from Cell Signalling Technology (Danvers, Massachusetts, United States).
LAT p200 mAb was obtained from Abcam (Cambridge, UK). Mouse anti-human antiphosphotyrosine (clone 4G10) mAb was from Millipore UK Ltd (Watford, UK). Alexa Fluor 488 phalloidin and Alexa Fluor 647 were purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). 1G5-Fab recognises monomeric and dimeric GPVI was raised as described 23 .
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