Osteoblast proteins were extracted using RIPA buffer including 1% protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and BCA assay was used to calculate the protein concentration. Then 30 µg proteins per lane was subjected to SDS-PAGE (10% gels) to separate, prior to their transfer onto PVDF membranes. After blocking with nonfat dry milk dissolved in TBS containing 0.05% Tween-20 for 1-2 h at room temperature, the membranes were incubated with antibodies against sirt1 (Santa Cruz Biotechnology, Inc.; cat. no. sc-74665); p53 (ProteinTech Group, Inc.; cat. no. 10442-1-AP) and p21 (ProteinTech Group, Inc.; cat. no. 28248-1-AP) and GAPDH (Santa Cruz Biotechnology, Inc.; cat. no. sc-137179) overnight at 4˚C at a dilution of 1:1,000. Subsequently, the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody for 1-2 h at room temperature at a dilution of 1:2,000, including goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc.; cat. no. sc-2004) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc.; cat. no. sc-2005). An enhanced chemiluminescence western blotting detection system (Santa Cruz Biotechnology, Inc.) and a GeneGnome HR scanner (Syngene) were used to visualize the immunoreactive proteins and chemiluminescent signal from the membranes. The expression levels of the proteins of interest were normalized to GAPDH.
Genegnome hr scanner
Manufactured by Syngene
The GeneGnome HR scanner is a high-resolution imaging system designed for genetic research applications. It provides accurate and sensitive detection of fluorescent signals in gel-based and microarray-based experiments. The scanner features a high-resolution imaging sensor and advanced optics to capture detailed images of DNA, RNA, and protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blotting detection system, by Santa Cruz Biotechnology (4 mentions)
Sirt1, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Pgc 1α, by Abcam (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Western blotting detection system, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Enhanced chemiluminescence western blotting detection system, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
6 protocols using genegnome hr scanner
1
Quantitative Analysis of Osteoblast Protein Markers
2
Western Blot Analysis of Osteoblast Proteins
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Osteoblastic MC3T3‐E1 cells were lysed using cold RIPA (Beyotime) containing 1% Protease Inhibitor Cocktail (Thermo Fisher) to obtain total protein. A 10% SDS‐PAGE was performed to separate the proteins, which were transferred to PVDF membranes. After blocking with 5% skimmed milk and 0.1% TBST for 2 H, the membranes were incubated with Sirt1 (Santa Cruz), PGC1α (Abcam), or β‐actin (Santa Cruz) antibodies in antibody dilution buffer (Beyome) at 4 °C overnight. The membranes were incubated with a secondary horseradish peroxidase‐conjugated antibody for 1–2 H at room temperature. The enhanced chemiluminescence Western blotting detection system (Santa Cruz) and a GeneGnome HR scanner (SynGene) were used to visualize the immunoreactive proteins and chemiluminescent signal from the membranes, respectively.
3
Western Blot Analysis of NKRF Protein
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The proteins of the H9c2 cardiomyocytes were lysed with cold RIPA lysis buffer (Beyotime). The protein load was 30 µg/lane in 10% SDS-PAGE, and it was subsequently transferred to nitrocellulose membranes. The bolts were blocked with 5% skim milk powder in 0.1%tris-buffered saline/Tween20 for 2 h and incubated with antibodies (Santa Cruz, Cat. No. sc-365568) against NKRF overnight at 4°C at a dilution of 1:1,000. Then, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. The immunoreactive proteins were visualized using the enhanced chemiluminescence western blotting detection system (Santa Cruz). The chemiluminescent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensities to β-actin was obtained to quantify the relative protein expression level.
4
Quantitative Western Blot Analysis of Mouse Renal Proteins
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Mouse renal tissue protein was lysed with chilled RIPA (Beyotime) with 1% Protease Inhibitor Cocktail (Sigma-Aldrich). We used 10% SDS-PAGE to separate protein, which was then transferred to NC membranes. The membrane was blocked with 5% skim milk powder in Tris-buffered saline containing 0.05% Tween 20 (TBST), then incubated with primary antibodies against PGC1α (Abcam), Sirt1 (Santa Cruz), or GAPDH (Santa Cruz), then incubated with secondary antibodies in milk. Chemiluminescence was performed with a Western blotting detection system (Millipore). The chemiluminescent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene).
5
Western Blot Analysis of Cysteine Metabolism Enzymes
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Tissue samples were lyzed with cold RIPA lysis buffer (Beyotime, China) and followed by centrifuging at 12,000 × g for 15min at 4°C. The supernatants were then collected. The protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, United States). Then, tissue extracts were mixed with 4× loading buffer containing 250mmol/L Tris-HCl, 10%SDS, 0.5% bromophenol blue, 50% glycerol and 7.5% DTT at pH 6.8. Samples were heated to 99°C for 10min before loading on a gel. The samples were separated by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes (Millipore Corp, Bedford, MA). The membranes were incubated with blocking buffer (Tris-buffered saline containing 0.1% Tween-20 and 5% skimmed milk powder) for 2h at room temperature, and then incubated with primary antibodies against CSE (Santa Cruz Biotech.; Cat# sc-365381), CBS (Santa Cruz Biotech.; Cat# sc-133208) or β-actin (Abcam; Cat# ab-8226) at 4°C overnight. After incubation with a secondary horseradish peroxidase-conjugated IgG (Santa Cruz) for 1h at room temperature, immunoblots were visualized using the enhanced chemiluminescence Western blotting detection system (Millipore). The chemiluminiscent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene).
6
Western Blot Analysis of Adrenal Proteins
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Mouse adrenal glands were lysed with cold RIPA lysis buffer (Beyotime). Protein load was 30 μg/lane in 10% SDS-PAGE subsequently transferred to nitrocellulose membranes. The bolts were blocked with 5% skim milk powder in 0.1% Tris-buffered saline/ Tween 20 (TBST) for 2h and incubated with antibodies against iNOS (Santa Cruz), SIRT1 (Santa Cruz) or ACTH receptor (Santa Cruz) overnight at 4˚C at a dilution of 1:1,000. Then, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence western blotting detection system (Santa Cruz). The chemiluminiscent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensities to the β-actin was obtained to quantify the relative protein expression level.
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