Cells were lysed in lysis buffer (1% NP‐40, 150 mM NaCl, 20 mM Tris‐HCl, pH 7.5, 0.5 mM EGTA, and 0.1 mM DTT) containing cOmplete mini protease inhibitor cocktail tablets (Merck) and phosphatase inhibitor cocktail (Nacalai Tesque). Cell lysates were boiled for 10 minutes with 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific). Proteins were separated using the NuPAGE SDS gel system (Thermo Fisher Scientific), electroblotted onto a PVDF membrane (Amersham Hybond‐P; Cytiva) and subjected to immunodetection using the following primary Abs at 1:1000 dilution; monoclonal mouse antibodies: anti‐α‐tubulin B‐5‐1‐2 (T5168; Merck), anti‐Cyclin B (610219; BD Biosciences), and anti‐Bcl‐2 (sc‐509; Santa Cruz Biotechnology); polyclonal rabbit antibodies: anti‐CHAMP1 (HPA008900; Atlas Antibodies), anti‐Mcl‐1 (4572; Cell Signaling Technology), anti‐Bcl‐xL (2762; Cell Signaling Technology), anti‐MAD2L2 (12683‐1‐AP; Proteintech), and anti‐Bak (3814; Cell Signaling Technology); and monoclonal rabbit antibody: anti‐Bim (2933; Cell Signaling Technology). Blocking and Ab incubations were carried out in 3% nonfat dry milk. Proteins were visualized using HRP‐labelled secondary Abs (1:5000; Santa Cruz Biotechnology) and enhanced chemiluminescence using ECL Prime Western Blotting Detection Reagents (Cytiva), according to the manufacturer’s instructions.
Amersham hybond p
Manufactured by Cytiva
Amersham Hybond P is a nitrocellulose membrane used for the immobilization and detection of proteins in Western blotting applications. It provides a stable and efficient support for the transfer and subsequent analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Benzonase nuclease, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage sds gel system, by Thermo Fisher Scientific (1 mentions)
Anti bcl2 sc 509, by Santa Cruz Biotechnology (1 mentions)
Complete mini protease inhibitor cocktail tablet, by Merck Group (1 mentions)
Anti mcl 1, by Cell Signaling Technology (1 mentions)
Anti bim, by Cell Signaling Technology (1 mentions)
Anti bak, by Cell Signaling Technology (1 mentions)
Anti bcl xl, by Cell Signaling Technology (1 mentions)
Anti α tubulin b 5 1 2, by Merck Group (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Odyssey clx infrared imager, by LI COR (1 mentions)
Irdye conjugated secondary antibody, by LI COR (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Trans blot sd, by Bio-Rad (1 mentions)
Ecl advance western blotting detection kit, by Cytiva (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
5 protocols using amersham hybond p
1
Western Blot Analysis of Cell Signaling Proteins
2
Immunoblotting Protocol with Detailed Antibody Information
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Details of the primary and secondary antibodies used for immunoblotting are provided in Supplementary Table 3 . For immunoblotting, total cell lysates were collected in 2× SDS–PAGE buffer and treated with benzonase nuclease (Millipore-Sigma). Proteins were separated on SDS–PAGE gels and transferred to polyvinylidenedifluoride membrane (Amersham Hybond P, Cytiva). Membranes were blocked for 30 min with 5% w/v milk powder (BioShop) in TBS, then incubated with primary antibodies in 5% w/v milk powder-TBST (TBS, 0.1% Tween-20) at 4 °C overnight. Following washing in TBST, blots were incubated with IRDye-conjugated secondary antibodies (LI-COR) in the dark for 1 h at room temperature. Blots were then washed three times in TBST and once in TBS, before imaging on a LI-COR Odyssey CLx Infrared Imager. Unprocessed blots are presented as source data.
3
Western Blotting of Testis Lysates
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Testes were lysed in RIPA buffer (Pierce) plus HALT protease inhibitor (Thermo Fisher Scientific), homogenized with an electronic pestle for 1 min, incubated at 4°C with agitation for 30 min, sonicated for 3 × 30 s, and then clarified at 14,000 × g at 4°C for 20 min. RPE lysates were collected in 2× SDS–polyacrylamide gel electrophoresis (PAGE) buffer and treated with benzonase nuclease (Millipore-Sigma) for 5 min. Samples were loaded into NuPAGE precast gels, transferred onto polyvinylidene fluoride (PVDF) membrane (Amersham Hybond P, Cytiva), and then rinsed in water then TBST, and then blocked in 5% milk in TBS plus 0.1% Tween. Membranes were then incubated overnight at 4°C in primary antibodies (Supplementary file 3 ) diluted in 5% milk TBST. Membranes were then washed 3 × 10 min TBST, incubated in Horse Radish Peroxidase (HRP)-conjugated secondary antibodies detailed in Supplementary file 4 for 1 hr at room temperature and developed using Pierce SuperSignal Pico Plus (Pierce) or ECL (GE Healthcare) reagent and imaged on ImageQuant.
4
Western Blot Analysis of TRPC6 Protein
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Western blot method was used to determine the changes in TRPC6 protein expression. The heart tissue (AAR of the left ventricle) was lysed and homogenized in RIPA Buffer (RIPA Lysis Buffer System, Santa Cruz Biotechnology; Wise Tis HG-15D Homogenizer). The total protein concentrations were determined by Bradford method and equal amount of protein samples was loaded in electrophoresis (Bio-Rad). Then, Protein samples were separated using 10% sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF; Amersham Hybond P) by Trans-Blot SD (Bio-Rad Semi-Dry Transfer Cell). The PVDF membranes were then incubated with primary TRPC6 antibody ((TRPC6 Polyclonal Antibody, Thermo Fisher Scientific, USA) and β-actin antibody as a loading control (Cell signaling Technology) followed by HRP-conjugated secondary antibody (Goat Anti Rabbit, Abcam). Eventually, The membrane was incubated with an ECL Western blotting system (Amersham ECL advance western blotting detection kit) and then exposed to X-ray film. Densitometry analysis of protein bands was performed using Image J software.
5
O-GlcNAcylation Quantification Assay
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The assay conditions were varied during the method development. Here, the final established assay is described. The O-GlcNAcylation reactions comprised: 10 μg/mL OGT, 25 U/ml alkaline phosphatase, 5 mM Mg-acetate, and 0.2 mg/mL OGT-substrate peptide-BSA complex (concentration based on BSA content), and 50 mM Bis-Tris pH 7.0 (adjusted with HCl). The reactions were carried out in 10 μL volume for 2h at room temperature (21-23°C). The sample volume comprised 2 μL of the reaction mixture. The reactions were stopped by dropping the temperature to 0°C. Bio-Rad microfiltration blotting device was employed to capture the OGT-substrate peptide-BSA complex onto 0.2 μm PVDF membrane (Amersham Hybond P). After completion of the dot blot, the membrane was completely dried, soaked in MeOH, rehydrated, and blocked with 0.7% Na-Caseinate pH 7.4–7.6, 1% BSA for 1h. O-GlcNAcylated residues were detected with the mouse monoclonal RL2 antibody (0.5 μg/mL, 2h), peroxidase-conjugated secondary antibody, and enhanced chemiluminescence.
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