Cells were lysed using RIPA lysis buffer with phenyl-methanesulfonyl fluoride (Wuhan Boster Biological Technology, Ltd.), and the protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). A total of 50 µg protein was loaded on a 10% SDS gel and resolved using SDS-PAGE. The resolved proteins were transferred to PVDF membranes. Non-specific binding sites were blocked by incubating the membranes with 5% non-fat milk, after which the membranes were incubated overnight at 4°C with the primary antibodies: ERα (1:1,000); HER2 (1:1,000); AKT (1:1,000); p-AKT (1:1,000); c-MYC (1:1,000); cyclin D1 (1:1,000); vimentin (1:1,000); E-cadherin (1:1,000); p21 (1:500); β-catenin (1:500); GAPDH (1:1,000) Membranes were subsequently incubated with a horseradish peroxidase-conjugated anti-rabbit/mouse immunoglobulin G secondary antibody (1:1,000; cat. no. BA1075 and BA1051; Wuhan Boster Biological Technology, Ltd.). Signals were visualized using enhanced chemiluminescence reagent (EMD Millipore). Densitometry analysis was performed using ImageJ and normalized to the respective expression of GAPDH.
Ripa lysis buffer
Manufactured by Wuhan Boster Biological Technology
RIPA lysis buffer is a common laboratory reagent used for extracting and solubilizing proteins from cells and tissues. It contains a mix of ionic and non-ionic detergents, as well as other components, to disrupt cell membranes and facilitate the release of proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Phenyl methanesulfonyl fluoride, by Wuhan Boster Biological Technology (1 mentions)
Ba1075, by Wuhan Boster Biological Technology (1 mentions)
Ba1051, by Wuhan Boster Biological Technology (1 mentions)
Chrebp, by Abcam (1 mentions)
Phosphorylated ampk thr 172, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Gapdh, by Wuhan Boster Biological Technology (1 mentions)
Ab2916, by Abcam (1 mentions)
Ab38949, by Abcam (1 mentions)
Ab7217, by Abcam (1 mentions)
Ab125219, by Abcam (1 mentions)
Ab59195, by Abcam (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
7 protocols using ripa lysis buffer
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of Lipogenic Proteins
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Protein was extracted using RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein (20 µg) was separated by 8% SDS-PAGE and transferred on to PVDF membrane. The membranes were incubated with antibodies of ChREBP (1:1,000, Abcam, cat. no. ab92809), fatty acid synthase (FASN; 1:500, Wuhan Boster Biological Technology, Ltd., cat. no. PB0909), acetyl-CoA carboxylase (ACC; 1:500, Wuhan Boster Biological Technology, Ltd., cat. no. BM4414), AMPK (1:1,000, Cell Signaling Technology, Inc., cat. no. 2532), phosphorylated (p)-AMPK (Thr172; 1:1,000, Cell Signaling Technology, Inc., cat. no. 2535), AKT (1:1,000, Cell Signaling Technology, Inc., cat. no. 9272), p-AKT (Ser473; 1:1,000, Cell Signaling Technology, Inc., cat. no. 9271), GAPDH (1:2,000, Wuhan Boster Biological Technology, Ltd., cat. no. BM1623) and FOXO1 (1:500, Wuhan Boster Biological Technology, Ltd., BM4249) overnight at 4°C. Then, membranes were incubated with rabbit anti-mouse lgG (cat. no. BA1048) and goat anti-rabbit lgG antibodies (cat. no. BA1039; both from Wuhan Boster Biological Technology, Ltd.). Finally, the results were detected by ECL reagents (Beyotime Institute of Biotechnology) and semi-quantified by densitometry using the Tanon 5200 automatic chemiluminescent imaging system (Tanon Science and Technology Co., Ltd).
3
Protein Expression Analysis in Colon Tissue
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Total protein was extracted from colon tissue and HT-29 cells using ice-cold RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.), and the protein concentrations were measured using a BCA protein assay kit (Boster). Equal amounts (20 µg) of protein was separated with 10% SDS-PAGE gel, then transferred onto PVDF membranes (EMD Millipore), and then blocked with 5% skim milk in TBST for 1 h at room temperature, and the membranes were incubated at 4°C overnight with primary antibodies against IKBα (ab7217; 1 : 2,000), p-p65 (ab86299; 1 : 5,000), p-IKK-β (ab59195), AQP3 (ab125219; 1 : 2,000), CFTR (ab2916; 1 : 2,000), PKA (ab38949; 1 : 2,000), and β-actin (ab8227; 1 : 1,000) all from Abcam (CA, USA). Then, the membranes were incubated with HRP-conjugated goat anti-rabbit immunoglobulin G (ab2057184; 1 : 5,000) for 1 h at room temperature. Proteins were visualized with the ECL western blotting detection reagents (Millipore). β-Actin was used as a loading control.
4
Western Blot Analysis of RAD54B
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Harvested hepatoma cells were washed with phosphate-buffered saline (PBS) and lysed using a RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.). The concentrations of protein were detected using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of total protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk-TBST and incubated with primary antibodies against RAD54B (1:500; Abcam) at 4°C overnight. The membranes were washed three times using TBST before being incubated with HRP-conjugated secondary antibodies (dilution 1:2000; cat. no. ab6721; Abcam) at room temperature for 1 h. The membranes were then washed three times with TBST and proteins were visualized using an electrochemiluminescence (ECL) assay. Images were taken by fusion-capture software (Fusion FX7; Vilber Lourmat, Marne Le-Vallée, France).
5
Ovarian Protein Expression Analysis
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Rats were anesthetized (5 mg/100 g, intraperitoneal injection) and euthanized by cervical dislocation on day 35, and bilateral ovaries were collected for subsequent experiments. Ovarian tissues were treated with RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, PRC). The protein concentration of each sample was determined with a BCA protein quantification kit (Wuhan Boster Biological Technology, Ltd.). Membranes were blocked with 5% dried skimmed milk (Wuhan Boster Biological Technology, Ltd.). The antibodies used in the experiment mainly include rabbit polyclonal anti-NGF (1 : 1000), rabbit polyclonal anti-FSHR (1 : 500), rabbit polyclonal anti-TrkA (1 : 500), and rabbit polyclonal anti-p75 (1 : 500; all Abcam, Cambridge, UK), goat anti-rabbit IgG secondary antibody (1 : 5000, Thermo Fisher Scientific, Inc.). The protein contents in the sample were calibrated and quantified with GAPDH (1 : 1000, Abcam) as internal reference. Immunodetection was performed using SuperSignal™ West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The X-ray films were developed, images were captured, and Quantity One software (Bio-Rad, Hercules, CA, USA) was used to analyze the gray values. The specific method of this experiment is shown in our previous research [13 (link)].
6
Western Blot Analysis of Protein Expression
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Total protein was extracted from PC cells using RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.), and the protein concentration was quantified using a bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology, Ltd.). Proteins (30 µg per lane) were loaded on a 10% gel and resolved using SDS-PAGE and transferred to PVDF membranes (EMD Millipore). Membranes were blocked using 5% skimmed milk, and subsequently incubated overnight using primary antibodies against target proteins, FOXO3 (cat. no. 10849-1-AP), β-catenin (cat. no. 51067-2-AP), TCF4 (cat. no. 22337-1-AP), E-cadherin (cat. no. 20874-1-AP), N-cadherin (cat. no. 22018-1-AP), vimentin (cat. no. 10366-1-AP) and GAPDH (cat. no. 60004-1-Ig) (all 1:1,000; ProteinTech Group, Inc.) at 4°C. Subsequently, the membranes were washed using TBS-Tween twice, and subsequently the membranes were incubated with horseradish peroxidase-conjugated anti-mouse (cat. no. BA1051) and anti-rabbit (cat. no. BA1055) secondary antibody (all 1:2,500; Wuhan Boster Biological Technology, Ltd.) and signals were visualized using enhanced chemiluminescent reagent (Wuhan Boster Biological Technology, Ltd.). Image Pro-Plus software was used to analyze the expression of protein, while GAPDH was used as a loading control.
7
Western Blot Analysis of EMT Markers
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Cells were washed with cold PBS and lysed with RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.). The protein concentration was determined by the BCA method. Protein (30 µg) was subjected to SDS-PAGE on a 10% gel and transferred onto a nitrocellulose membrane (GE Healthcare). Following blocking with 5% non-fat milk for 1 h at room temperature, the membrane was incubated at 4°C overnight with primary antibodies against each of the following proteins: E-cadherin, β-catenin, vimentin, Twist1 and GAPDH (all 1:1,000 dilution). Following washing, the membrane was incubated with corresponding horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution) for 1 h at room temperature. The membrane was washed and immersed in SuperSignal West Pico Chemiluminescent Substrate detection kit (Thermo Fisher Scientific, Inc.). Bound secondary antibody was detected by the Amersham™ Imager 600 system (GE Healthcare Bio-Sciences). Image J software v1.8.0.112 (National Institutes of Health) was used to quantify all protein band densities. The level of the tested proteins is represented by determining the ratio of the integrated density of tested protein relative to that of the GAPDH loading control.
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