beta Tubulin Monoclonal Antibody (2 28 33)

Protocol tips

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	This procedure is based on a description reported previously (Vega et al. 2011). In brief, C2C12 myotubes were washed twice with PBS and then exposed to RIPA buffer consisting of (mm): 20 Tris‐HCl (pH 7.5), 150 NaCl, 1 Na2EDTA, 1 EGTA, 10 NaPyroPO4, 10 β‐glycerophosphate, 10 NaF and 1 Na3VO4; supplemented with 1% NP‐40, 1% sodium deoxycholate and a proteinase inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Subsequently, protein samples (20–50 μg) were subjected to SDS‐PAGE, transferred to polyvinylidene difluoride membranes and analysed by western blotting. The primary antibodies and dilutions used were: anti‐RyR1 (ARR‐001; Alomone Labs, Jerusalem, Israel; dilution 1:1000), anti‐P‐PBL (07‐052; Upstate Biotechnology, Lake Placid, NY, USA; dilution 1:1500), anti‐STIM1 (ACC‐063; Alomone; dilution 1:1000) and anti‐Orai1 (ACC‐062; Alomone; dilution 1:500). Each particular membrane was also probed with an anti‐β‐tubulin antibody (32‐2600; Invitrogen; dilution 1:4000) to normalize for the amount of protein loaded. Immunoreactivity was revealed by secondary antibodies coupled to peroxidase (either 32 260; Pierce Biotechnology, Rockford, IL, USA; dilution 1:15,000 or G‐21040; Invitrogen; dilution 1:10,000). Band analysis was performed with ImageJ (NIH, Bethesda, MD, USA).

Protocol tips

This procedure is based on a description reported previously (Vega et al. 2011). In brief, C2C12 myotubes were washed twice with PBS and then exposed to RIPA buffer consisting of (mm): 20 Tris‐HCl (pH 7.5), 150 NaCl, 1 Na2EDTA, 1 EGTA, 10 NaPyroPO4, 10 β‐glycerophosphate, 10 NaF and 1 Na3VO4; supplemented with 1% NP‐40, 1% sodium deoxycholate and a proteinase inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Subsequently, protein samples (20–50 μg) were subjected to SDS‐PAGE, transferred to polyvinylidene difluoride membranes and analysed by western blotting. The primary antibodies and dilutions used were: anti‐RyR1 (ARR‐001; Alomone Labs, Jerusalem, Israel; dilution 1:1000), anti‐P‐PBL (07‐052; Upstate Biotechnology, Lake Placid, NY, USA; dilution 1:1500), anti‐STIM1 (ACC‐063; Alomone; dilution 1:1000) and anti‐Orai1 (ACC‐062; Alomone; dilution 1:500). Each particular membrane was also probed with an anti‐β‐tubulin antibody (32‐2600; Invitrogen; dilution 1:4000) to normalize for the amount of protein loaded. Immunoreactivity was revealed by secondary antibodies coupled to peroxidase (either 32 260; Pierce Biotechnology, Rockford, IL, USA; dilution 1:15,000 or G‐21040; Invitrogen; dilution 1:10,000). Band analysis was performed with ImageJ (NIH, Bethesda, MD, USA).

Publication protocol

This procedure is based on a description reported previously (Vega et al. 2011). In brief, C2C12 myotubes were washed twice with PBS and then exposed to RIPA buffer consisting of (mm): 20 Tris‐HCl (pH 7.5), 150 NaCl, 1 Na2EDTA, 1 EGTA, 10 NaPyroPO4, 10 β‐glycerophosphate, 10 NaF and 1 Na3VO4; supplemented with 1% NP‐40, 1% sodium deoxycholate and a proteinase inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Subsequently, protein samples (20–50 μg) were subjected to SDS‐PAGE, transferred to polyvinylidene difluoride membranes and analysed by western blotting. The primary antibodies and dilutions used were: anti‐RyR1 (ARR‐001; Alomone Labs, Jerusalem, Israel; dilution 1:1000), anti‐P‐PBL (07‐052; Upstate Biotechnology, Lake Placid, NY, USA; dilution 1:1500), anti‐STIM1 (ACC‐063; Alomone; dilution 1:1000) and anti‐Orai1 (ACC‐062; Alomone; dilution 1:500). Each particular membrane was also probed with an anti‐β‐tubulin antibody (32‐2600; Invitrogen; dilution 1:4000) to normalize for the amount of protein loaded. Immunoreactivity was revealed by secondary antibodies coupled to peroxidase (either 32 260; Pierce Biotechnology, Rockford, IL, USA; dilution 1:15,000 or G‐21040; Invitrogen; dilution 1:10,000). Band analysis was performed with ImageJ (NIH, Bethesda, MD, USA).

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