Anti-beta Tubulin antibody - Loading Control (ab6046)

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	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.).

Publication protocol

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.).

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