Prior to CD44 analysis by western blot the surface proteins were isolated (Pierce® Cell surface protein isolation kit, Pierce Biotechnology, Rockford, IL, USA). Molecular weight separation was performed on 10% Tris-HCl gels with Tris/glycine/SDS buffer, the proteins transferred to nitrocellulose membranes, and CD44 detected with sheep anti-rat CD44 (0.1 µg/mL; R&D Systems, Minneapolis, MN, USA) and HRP-conjugated rabbit anti-sheep (1:5000; Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Luminescent signal was captured on an enhanced chemiluminescence (ECL)-camera system (Kodak image station 2000; New Haven, CT, USA). β-actin was detected with mouse anti-rat β-actin antibody (1:20,000, Sigma-Aldrich, St Louis, MO, USA) and secondary horseradish peroxidase (HRP)-conjugated goat-anti mouse antibody (1:10,000; Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). CD44 western blot analysis of samples from isolated surface proteins was normalized to the β-actin expression.
Image station 2000
Manufactured by Kodak
Sourced in United States
The Image Station 2000 is a digital imaging system designed for laboratory use. It provides high-quality image capture and processing capabilities. The product's core function is to digitize and store photographic images, enabling efficient image management and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti rat β actin antibody, by Merck Group (1 mentions)
Pierce cell surface protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Rabbit anti enos, by Merck Group (1 mentions)
Complete mini, by Roche (1 mentions)
4 protocols using image station 2000
1
CD44 Expression Analysis by Western Blot
2
Immunoblotting Protein Detection Protocol
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Colon homogenates or cell lysates (30 μg) were resolved by 7.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred electrophoretically to PVDF membranes. The membranes were incubated for 1 h with 5% bovine serum albumin to block non-specific binding sites and then with primary antibodies at 4 °C for overnight. Thereafter, the membranes were incubated with the secondary antibody conjugated with horseradish peroxidase, which was detected with the chemiluminescent detection system (Vazyme, Nanjing, China; Cat: E412-01). The chemiluminescent signal was captured by Kodak Image Station 2000.
3
Western Blot Analysis of Signaling Proteins
4
Immunoblotting for Neuronal and Endothelial NOS
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Isolated PTC were mixed 1:1 with lysis buffer (1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 80 mM Tris; pH 7.5) containing enzyme inhibitors (Complete Mini; 1 tablet/3 mL; Roche Diagnostics, Mannheim, Germany). Total homogenates from rat brain were used as positive controls. Samples were run on 7.5% Tris-HCl gels with Tris/glycine/SDS buffer. Proteins were detected, after transfer to nitrocellulose membranes, using rabbit anti-nNOS (2 μg/mL; Zymed Laboratories, Invitrogen, Carlsbad, CA, USA), rabbit anti-eNOS (1:2000; Sigma Aldrich), and HRP-conjugated secondary goat anti-rabbit antibody (1:5000; Sigma-Aldrich) by an ECL-camera (Kodak image station 2000; New Haven, CT, USA).
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