Western blotting Tubulin beta

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beta Tubulin Monoclonal Antibody (2 28 33)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream 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).

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).

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).

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-beta Tubulin antibody - Loading Control (ab6046)

Abcam

Upstream tips	Protocol tips	Downstream tips
	cell lysates were collected in supplemented 1% NP‐40 (Sigma‐Aldrich) and total protein content in cell lysate was quantified with BCA protein assay kit (Thermo Scientific). Collected lysates were then reduced, and size separated protein samples were transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was incubated for 2 h with α‐SMA antibody (0.3–1 μg/mL, ab5694, Abcam, Cambridge, UK), along with β‐tubulin antibody (1:30,000–40,000, ab6046, Abcam) or GAPDH antibody (0.2 μg/mL, sc‐47724, Santa Cruz) in blocking buffer (0.5% casein in 0.5% TBS‐Tween), before washing in TBS‐Tween, and incubation (30 min, RT) with a secondary DyLight680 or DyLight800‐conjugated antibody, prior to final washing steps. Core protein bands of α‐SMA and endogenous controls (GAPDH and β‐tubulin) were imaged with Odyssey FC (LI‐COR Inc., Lincoln, NE), controlled and analyzed by Image Studio v.3.1 (LI‐COR Inc.).

Protocol tips
cell lysates were collected in supplemented 1% NP‐40 (Sigma‐Aldrich) and total protein content in cell lysate was quantified with BCA protein assay kit (Thermo Scientific). Collected lysates were then reduced, and size separated protein samples were transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was incubated for 2 h with α‐SMA antibody (0.3–1 μg/mL, ab5694, Abcam, Cambridge, UK), along with β‐tubulin antibody (1:30,000–40,000, ab6046, Abcam) or GAPDH antibody (0.2 μg/mL, sc‐47724, Santa Cruz) in blocking buffer (0.5% casein in 0.5% TBS‐Tween), before washing in TBS‐Tween, and incubation (30 min, RT) with a secondary DyLight680 or DyLight800‐conjugated antibody, prior to final washing steps. Core protein bands of α‐SMA and endogenous controls (GAPDH and β‐tubulin) were imaged with Odyssey FC (LI‐COR Inc., Lincoln, NE), controlled and analyzed by Image Studio v.3.1 (LI‐COR Inc.).

Protocol tips

cell lysates were collected in supplemented 1% NP‐40 (Sigma‐Aldrich) and total protein content in cell lysate was quantified with BCA protein assay kit (Thermo Scientific). Collected lysates were then reduced, and size separated protein samples were transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was incubated for 2 h with α‐SMA antibody (0.3–1 μg/mL, ab5694, Abcam, Cambridge, UK), along with β‐tubulin antibody (1:30,000–40,000, ab6046, Abcam) or GAPDH antibody (0.2 μg/mL, sc‐47724, Santa Cruz) in blocking buffer (0.5% casein in 0.5% TBS‐Tween), before washing in TBS‐Tween, and incubation (30 min, RT) with a secondary DyLight680 or DyLight800‐conjugated antibody, prior to final washing steps. Core protein bands of α‐SMA and endogenous controls (GAPDH and β‐tubulin) were imaged with Odyssey FC (LI‐COR Inc., Lincoln, NE), controlled and analyzed by Image Studio v.3.1 (LI‐COR Inc.).

Manufacturer protocol Publication protocol 1 Relevant paper

β Tubulin Antibody (TUB 2.1): sc-58886

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Immunoblotting was performed as previously described [51].

Protocol tips
Immunoblotting was performed as previously described [51].

Manufacturer protocol Publication protocol 1 Relevant paper

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