HFSCs were starved and then treated with 300 ng/ml of Wnt5a for 2 h prior to harvesting for small GTPase activation assay. For small GTPase activation assay, Rac1/Cdc42 activation assay combo kit (Cell Biolabs) was performed according to the manufacturer protocol. For immunoblotting analysis, proteins were extracted from cells with RIPA buffer (50 mM Tris-HCl, 100 mM NaCl, 1% of NP-40, 0.5% of NaDeoxycholate, 0.1% of SDS, 1 mM EDTA, protease inhibitor, phosphatase inhibitors). Protein lysates were then loaded into a 4–12% Bis-Tris gel (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific). Transferred membranes were blocked with 5% BSA in TBST buffer at RT for 1 h, and then incubated with primary antibodies at 4 °C overnight. After incubation, blots were washed and then incubated with the HRP-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h. The blots were developed using chemiluminescence detection reagent (SuperSignal West substrates, Thermo Fisher Scientific), and signals were then detected using the Fusion SL imaging system (Vilber) with Fusion-Capt Advance Solo 4S v16.16b software.
Supersignal west substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal West Substrate is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It generates a luminescent signal upon reaction with the horseradish peroxidase (HRP) enzyme, which is commonly used to label antibodies. The intensity of the luminescent signal is proportional to the amount of the target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (10 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (6 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions)
Runx2, by Cell Signaling Technology (3 mentions)
Versadoc imaging system, by Bio-Rad (3 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions)
Radioimmunoprecipitation assay rapi buffer, by Thermo Fisher Scientific (2 mentions)
Nupage sample reducing reagent, by Thermo Fisher Scientific (2 mentions)
Novex mini cell device, by Thermo Fisher Scientific (2 mentions)
Anti cyclin d1 primary antibody, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Anti ki67, by Cell Signaling Technology (2 mentions)
Sds page gel, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
27 protocols using supersignal west substrate
1
Wnt5a-Induced GTPase Activation Assay
2
Western Blot Protein Extraction and Analysis
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Cells were washed with phosphate-buffered saline (PBS, pH7.4) and whole-cell proteins were extracted by 1x RIPA lysis buffer (Cat# 9806, Cell Signaling Tech, MA) plus proteinase inhibitor cocktail (Cat# 4693132001, Roche, MilliporeSigma, MA) and phosphatase inhibitor cocktail (Cat# 4906837001 PHOSS-RO, MilliporeSigma). The cell lysates were sonicated and centrifuged to remove debris. IB analysis was performed following standard IB protocols described in our previous publications (13, (link)23, (link)24) (link). Briefly, equal amounts of total proteins were loaded and separated by precast gels (Cat# 4561086, Bio-Rad, CA; or Cat# M00657, GenScript, NJ) and transferred to Immun-Blot ® PVDF membranes (Cat# 1620177, Bio-Rad). The membranes were blocked by nonfat milk and incubated with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies (goat anti-rabbit HRP, Cat# 31460 and goat anti-mouse HRP, Cat# 31430, Thermo Fisher Scientific). The protein blotting signals were visualized by incubating the membranes with SuperSignal ™ West substrates (Cat# 34580, Cat# 37071, or Cat# 34096, Thermo Fisher Scientific) and scanned by a VersaDoc ™ MP Image System (Bio-Rad). The protein blotting images were obtained, processed, and quantified by Image Lab ™ (Ver. 6.0.1 build 34, Bio-Rad). All antibodies used in IB analysis are listed in Supplementary Table S3.
3
Protein Purification and Western Blotting
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Total protein was purified using Radioimmunoprecipitation assay (RAPI) buffer (Thermo fisher scientific). Concentration of protein samples were determined by Pierce BCA protein assay kit (Thermo fisher scientific). Protein samples mixed with loading buffer and NuPAGE sample reducing reagent (Thermo fisher scientific) were heat-shocked at 85℃ for 2 minutes before loaded into SDS-PAGE gel (Thermo fisher scientific). Electrophoresis was performed on a Novex mini-cell device (Invitrogen, Carlsbad, CA, USA) for 90 munities. The gel was then attached to a PVDF membrane (Thermo fisher scientific) to transfer the protein. After blocked in 1% BSA buffer for 1 hour, the membrane was incubated in diluted antibodies solution overnight at 4 ℃. Anti-KPNB1, anti-Cyclin B1 primary antibodies were purchased from Novus biologicals (Littleton, CO, USA). Anti-Cyclin D1 primary antibody was purchased from Santa Cruz Biotechnology. Anti-CDK1, anti-RCC1, anti-pRCC1, anti-β-actin, anti-Ki67, and anti-caspase 3 primary antibodies and all secondary antibodies were purchased from Cell signaling technology (Danvers, MA, USA). Targeted protein was imaged using SuperSignal West substrate (Thermo fisher scientific).
4
Protein Expression Analysis in Osteogenesis
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Whole cell lysates and western blot analyses were performed as described previously44 (link). The primary antibodies used were as follows: Runx2 rabbit monoclonal antibody (1:2000) (Epitomics, Burlingame CA), anti-Sp7/Osterix rabbit polyclonal antibody (1:5000) (Abcam, UK), anti-KAT3B/p300 antibody (1:2000) (Abcam), and anti-acetyl lysine antibody (1:1000) (Abcam). Membranes were incubated for 1 h at room temperature with the primary antibody in 5% milk followed by another incubation with a horseradish peroxidase-conjugated secondary antibody. The signals were detected using the Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometry analyses of the western bands were performed using the Tanon Imaging software67 (link).
5
Protein Purification and Western Blotting
6
Liver Protein Expression Analysis
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Approximately 50 mg of frozen liver tissue was homogenized in 0.3 ml RIPA buffer containing 1 mM PMSF and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein concentration was determined via a BCA assay kit (Pierce; Therm Fisher Scientific, Inc.). The homogenate proteins (75 µg) from individual liver samples were resolved by SDS-PAGE (10% Criterion™ Tris-HCl Protein Gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto nitrocellulose membranes; membranes were blocked with 5% non-fat milk (cat. no. 170-6404; Bio-Rad Laboratories, Inc.). Anti-SR-BI antibody was purchased from Abcam (cat. no. Ab52629). Anti-β-actin antibody was purchased from Sigma-Aldrich; Merck KGaA (cat. no. A1978). Primary antibody was used at 1:1,000 dilution and secondary antibodies (cat. nos. 7074P2 and 7076P2) were used at 1:10,000 dilution. Immunoreactive bands of predicted molecular mass were visualized using SuperSignal West substrate (Thermo Fisher Scientific) and quantified using Alpha View software (version 3.3; Cell Biosciences, Inc., Santa Clara, CA, USA) with normalization by signals of β-actin.
7
Liver Protein Extraction and Western Blot Analysis
8
Western Blot Analysis of Osteogenic and Apoptotic Markers
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The total protein was extracted from MC3T3-E1 cells with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, USA) including a 10% protease inhibitor cocktail (Roche, Switzerland). The protein concentration was measured by a Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein samples containing loading buffer were subjected to electrophoresis on NuPAGE™ Bis–Tris Protein Gels (Invitrogen, USA) for 2 h. Proteins were transferred to polyvinylidene difluoride membranes, and the membranes were blocked in 5% skim milk for 4 h. Then, the PVDF membranes were incubated with primary antibodies specific for Runx2 (1:1000, Cell Signaling Technology, USA), Caspase-3 (1:1000, Cell Signaling Technology, USA), and β-actin (1:1000, Cell Signaling Technology, USA) overnight at 4 °C. The next day, after washing with TBST, the PVDF membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, ZSGB-BIO, China) for 1 h. The signals were visualized by Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometric analyses of the bands were performed using Tanon Imaging software.
9
Immunoblotting of Liver Protein Targets
10
Western Blot Analysis of Osteogenic and Exosomal Markers
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Proteins in femurs, exosomes, or MC3T3-E1 cells were collected in RIPA buffer (Thermo Scientific, USA). The same amount of protein samples was loaded onto NuPage Bis–Tris polyacrylamide gels (Invitrogen, USA) and then proteins were transferred onto polyvinylidene difluoride membranes. After blocking in milk (5% w/v) for 4 h at room temperature, the membranes were subsequently incubated overnight at 4 °C with primary antibodies against GAPDH (1:5000; Proteintech, USA), Runx2 (1:1000; Cell Signaling Technology, USA), Bglap (1:500; Abcam, USA), Col1a1 (1:1000; Abcam, US), CD81 (1:1000; Abcam, USA), and TSG101 (1:1000; Abcam, USA). Next, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000; Jackson, USA), and the signals were visualized using SuperSignal West substrate (Thermo Fisher Scientific, USA). The density was measured and analyzed using ImageJ software.
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