Cell lysate was added into the −80 °C frozen spinal cord tissue and rapidly homogenized on ice and centrifuged at 4 °C. The supernatant was aliquot and the concentration of protein was determined via the BAC method. Samples containing 80 μg of protein were loaded into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), proteins were then electrotransferred to a polyvinylidene fluoride (PVDF) membrane (Amersham Bio-sciences, Freiburg, Germany) and blocked at room temperature in a blocking solution consisting of TBST (Tris-buffer saline containing 0.05% Tween-20). Membranes were then incubated overnight at 4 °C with primary antibodies of anti-PKG1 (Cell Signaling Technology, Boston, MA, American, 1:1000) and anti-GAPDH (Sigma Chemical Co, St. Louis, MO, USA, 1:1000) dissolved in TBST containing 5% nonfat dry milk. Membranes were then washed with TBST and incubated with HRP labeled secondary antibody (ComWin Biotech, Beijing, China, 1:5000) diluted with 5% nonfat dry milk under room temperature for one hour. The membrane was detected by reaction with the Amersham ECL-Plus reagent (Amersham Bio-sciences, Freiburg, Germany).
Anti pkg1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-PKG1 is a primary antibody that specifically recognizes the protein kinase G1 (PKG1) enzyme. PKG1 is a serine/threonine-specific protein kinase that plays a crucial role in various cellular signaling pathways. The Anti-PKG1 antibody can be used to detect and quantify PKG1 expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Cytiva (1 mentions)
Hrp labeled secondary antibody, by CoWin Biotech (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Supersignal west pico chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by AbFrontier (1 mentions)
Anti vegfr1, by Cell Signaling Technology (1 mentions)
Anti pnfatc4, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti fus, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Anti nephrin, by Progen Biotechnik (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
5 protocols using anti pkg1
1
Western Blot Analysis of PKG1 in Spinal Cord
2
Western Blot Analysis of Cardiac Proteins
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Protein samples were prepared from NRVMs using 1% sodium dodecyl sulfate (SDS) lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche Diagnostics). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% BSA (Sigma-Aldrich) in TBST (0.1% Tween-20 in Tris-buffered saline: 137 mM NaCl and 20 mM Tris-HCl, pH 7.4) for 1 h at room temperature. For NFAT, 8% skim milk (Difco, Detroit, MI, USA) in TBST was used for blocking. Membranes were then incubated overnight at 4°C with the following primary antibodies: anti-VEGFR1, anti-PKG-1, anti-phospho (p)-CaMKII (Thr286) (Cell Signaling Technology, MA, USA), anti-p-NFATc3, anti-p-NFATc4, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Next, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (AbFrontier, Seoul, Korea) at room temperature for 1 h, and luminescence was detected using an ImageQuant LAS 4000 mini (GE Healthcare) and a SuperSignal West Pico Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA). The intensity of each protein band was quantified by NIH ImageJ software.
3
Western Blot Analysis of Protein Expression
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Total protein isolated from cells and tissues using RIPA lysis buffer (ProMab Biotechnology, USA). Equal amounts of protein were separated on 10% SDS polyacrylamide gels and then electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking in 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies including anti-EMX2OS (1:400; Abcam, Cambridge, UK), anti-TCF12 (1:400; Abcam), anti-FUS (1:400; Abcam), anti-PKG1 (1:500; Cell Signaling Technology, Boston, MA), anti-PKG2 (1:500; Cell Signaling Technology) and β-actin (1:600; Abcam). Next, the membranes were incubated with proper horseradish peroxidase (HRP)-conjugated IgG for 1 h at 37 °C. Protein bands were visualized using an enhanced chemiluminescence detection system (ECL; Beyotime, Shanghai, China).
4
Western Blot Analysis of Renal Proteins
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Renal homogenates were separated by SDS-PAGE electrophoresis, blotted on nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), and incubated with primary antibodies, followed by 1 h incubation with fluorescent secondary antibodies (Alexa Fluor, 680 nm). The Western blotting was analyzed and quantified by Odyssey Infrared Imaging Detection System (LICOR Biosciences, Lincoln, NE, USA). The optical densities were expressed as arbitrary units. Antibodies: anti-nephrin (Progen, Heidelberg, Germany); anti-actin (Sigma-Aldrich); anti-α1 Na-K ATPase (Hybridoma Bank, clone a6F, Iowa City, IA, USA); anti-PKG1 (Cell Signaling Technology, Danvers, MA, USA); anti-Bcl2 (Santa Cruz Biotechnology, CA, USA); anti-GAPDH (Abcam, Cambridge, UK).
5
Protein Expression Analysis in Mouse Cells
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Protein was isolated from mouse AoSMCs and organs, as previously described. 33 Protein (15 mg) was used and separated in an SDS-PAGE gel and transblotted. After blocking with 5% skim milk, blots were incubated with the primary antibodies: anti-PKG1 1:200 (Cell Signaling), anti-calponin 1:200 (Sigma), anti-osteopontin 1:200 (Abcam), and antiea-tubulin 1:1000 (Abcam), and antiglyceraldehyde-3-phosphate dehydrogenase 1:1000 (Abcam) as control. Appropriate secondary antibodies were then used, and the protein bands were detected using the ECL Prime Western Blotting Detection Reagent (GE Health Care, Little Chalfont, UK) and quantified with ImageJ software version 1.48v (NIH, Bethesda, MD).
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