Western blot analysis was performed as previously described by us [16 (link)]. Twenty µl of the cell/vessel extracts were loaded onto 10% Bis-Tris SDS-polyacrylamide gels (Thermo Fisher Scientific), separated for 35 min at 200 V and finally electro-transferred to nylon-PVDF Immobilon-PSQ membranes for 90 min at 30 V in 20% methanol blotting buffer. Briefly, blots were blocked for 30 min in blocking buffer, incubated with primary antibodies against PDGFRβ clone Y92 (C-terminal, amino acid (aa)1050-1150) (1:3000, Novus Biologicals NB110-57343), alpha-smooth muscle actin (1:1000, abcam ab119952), actin (1:1000, Sigma A2066) or laminin (1:500, Sigma) at 4°C overnight, washed, and then incubated in alkaline phosphatase conjugated anti-rabbit IgG or anti-mouse IgG for 30 min. After washing, bound antibodies were detected using an enhanced chemiluminescence (ECL) system and visualized by using a cooled CCD camera (SearchLight; Thermo Fisher Scientific).
Protocol detail
Western Blot Analysis of Cell Extracts
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Actin
Alkaline phosphatase
Alpha actin
Amino acid
Anti igg
Antibodies
Biologicals
Bis tris
Buffer
Cell extracts
Chemiluminescence
Clone
Immobilon
Laminin
Membranes
Methanol
Mouse
Novus
Nylon
Pdgfrβ
Polyacrylamide gels
Pvdf
Rabbit
Smooth muscle
Vessel
Western blot analysis
Corresponding Organization :
Other organizations : Innsbruck Medical University, Universität Innsbruck
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Variable analysis
independent variables
- Antibody concentration against PDGFRβ clone Y92 (C-terminal, amino acid (aa)1050-1150) (1:3000)
- Antibody concentration against alpha-smooth muscle actin (1:1000)
- Antibody concentration against actin (1:1000)
- Antibody concentration against laminin (1:500)
- PDGFRβ protein expression
- Alpha-smooth muscle actin protein expression
- Actin protein expression
- Laminin protein expression
- Cell/vessel extracts sample volume (20 µl)
- Gel type (10% Bis-Tris SDS-polyacrylamide gels)
- Separation time (35 min at 200 V)
- Electro-transfer time (90 min at 30 V)
- Blotting buffer (20% methanol)
- Blocking time (30 min)
- Incubation temperature (4°C overnight)
- Secondary antibody type (alkaline phosphatase conjugated anti-rabbit IgG or anti-mouse IgG)
- Secondary antibody incubation time (30 min)
- Detection method (enhanced chemiluminescence (ECL) system and CCD camera)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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