Testis and isolated Sertoli cells were prepared by three repeated freeze-thaw cycles in a lysis buffer containing (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 0.5% Igepal) and protease and phosphatase inhibitors (Sigma) as described in Froment (2004) (link). Proteins were submitted to electrophoresis on SDS-PAGE under reducing conditions. After transfer, the membranes were incubated overnight at 4°C with antibodies against phospho-(Thr172) AMPKα (New England Biolabs, Inc., Beverly, MA, USA), AMPKα1 (Upstate Biotechnology, Inc.), PCNA, phosphorylated (Ser473) protein kinase B (AKT), AKT, phosphorylated (Thr202/Tyr204) ERK1/2, cyclin D2 (Cell Signalling), ERK, P21 and P27 (Santa Cruz Biotechnology), and vinculin (VCL) (Sigma). All antibodies were used at 1:1000 dilution. As secondary antibodies, HRP-linked sheep anti-mouse IGG antibody or donkey anti-rabbit IgG antibody (1:10 000, Amersham Biosciences) was used. The signal was detected by ECL (Amersham Pharmacia) and quantified using Image Analysis Software (ImageJ, v 1.48, NIH) . The results are expressed as signal intensity in arbitrary units, after normalisation by an internal standard (total protein for phosphorylated proteins or vinculin) and correspond to the mean of three separate experiments.
Vcl vinculin
Manufactured by Merck Group
Sourced in United States
VCL/vinculin is a cytoskeletal protein that plays a key role in cell-cell and cell-matrix adhesion. It is involved in the linkage between the actin cytoskeleton and the extracellular matrix or adjacent cells. VCL/vinculin is a commonly used marker for studying cell adhesion and cytoskeletal organization in various cell types and tissues.
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Lab products found in correlation
Np0007, by Thermo Fisher Scientific (3 mentions)
Imagequant tl, by Cytiva (3 mentions)
Np0005, by Thermo Fisher Scientific (3 mentions)
β actin actb, by Merck Group (2 mentions)
Np0002, by Thermo Fisher Scientific (2 mentions)
Nxa931, by GE Healthcare (2 mentions)
Tuba4a tuba1a α tubulin, by Merck Group (2 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Fkbp5 fkbp5, by Cell Signaling Technology (1 mentions)
Fkbp5 fkbp51, by Cell Signaling Technology (1 mentions)
Pierce ecl, by Thermo Fisher Scientific (1 mentions)
Mab2166, by Merck Group (1 mentions)
Np00061, by Thermo Fisher Scientific (1 mentions)
5 protocols using vcl vinculin
1
Western Blot Analysis of Testis Proteins
2
Antibody Reagents for Western Blotting
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Rabbit polyclonal antibodies to APLN and APLNR were purchased from Antibodies-online and Sigma respectively. Rabbit polyclonal antibodies to phospho-MAPK ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-PRKA (Thr172) and total PRKA were obtained from Ozyme (Montigny Le Bretonneux, France). Mouse monoclonal antibodies to VINCULIN (VCL) were obtained from Sigma. Rabbit polyclonal antibodies recognizing total ERK2 (C14), phospho-AKT (Ser 473) and total MAPK P38 were purchased from Santa Cruz Biotechnology. All antibodies were used at 1:1000 for Western blotting.
3
Western Blotting Protocol for Protein Quantification
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Lysates (8–15 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. Lysates were run on a 4–12% Bis-tris gel at 200 V for 50 min in MES running buffer (Invitrogen, NP0002) with antioxidant (Invitrogen, NP0005). Transfer was performed ON, 20 V for 840 min at 4°C, onto a 0.4-μm nitrocellulose membrane. Blocking was done with 5% milk in TBST. After blocking membranes were probed with the following primaries: FKBP5/FKBP5 (1:100; Cell Signaling Technology, 12,210), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441), or TUBA4A/TUBA1A/α-tubulin (1:1000; MilliporeSigma, T6199). Membranes were incubated with secondary anti-murine HRP-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit HRP-coupled antibodies (1:2500; GE Healthcare, NA934) at RT for 2 h in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.
4
Western Blot for Protein Quantification
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Lysates (15–30 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. The R6/2 lysates were run on a 4–12% Bis-tris gel at 200 V for 50 min in MES running buffer (Invitrogen, NP0002) with antioxidant (Invitrogen, NP0005). The zQ175 lysates were separated on 3–8% tris-acetate gel at 200 V for 90 min in tris-acetate running buffer. Transfer was performed ON, 20 V for 840 min at 4°C, onto a 0.4-μm nitrocellulose membrane. Blocking was done with 5% milk in TBST. After blocking membranes were probed with the following primaries: HTT (1:500; MilliporeSigma, 5492), FKBP5/FKBP51 (1:100; Cell Signaling Technology, 12,210), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441), or TUBA4A/TUBA1A/α-tubulin (1:1000; MilliporeSigma,T6199). Membranes were incubated with secondary anti-murine HRP-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit (1:2500;GE Healthcare, NA934) at RT for 2 h or ON at 4°C in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.
5
Huntingtin Protein Immunoblotting Assay
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Lysates (20 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. mHTT and HTT bands were separated on a 3–8% Tris-acetate gel, run for 90 min in Tris-acetate running buffer (Invitrogen, LA0041) and antioxidant (Invitrogen, NP0005) at 200 V on ice. Transfer was performed at 4°C at a constant 20 V for 840 min in transfer buffer (Thermo Fischer Scientific, NP00061) onto a nitrocellulose membrane. Blocking was done ON in 5% milk in TBST, primary antibody HTT (1:250; Millipore MAB2166), HTT (1:500; Millipore, 1574), VCL/vinculin (1:500; MilliporeSigma, V9131) was probed at 4°C ON in 5% milk TBST, secondary HRP-coupled anti-mouse (GE Healthcare, NXA931; 1:2500) was probed ON at 4°C in 5% milk TBST. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.
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