The protein concentration of isolated extracellular vesicles in 300 µl was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). The samples were lysed with RIPA reagent (Millipore) containing a protease inhibitor cocktail (Roche, Mannheim, Germany) and 10% 0.1 M PMSF (phenylmethyl sulphonyl Fluoride, Sigma‐Aldrich). The proteins were denatured in SDS‐PAGE sample buffer by heating at 95°C for 15 min. Next, 40‐μg protein samples were subjected to 12% SDS‐PAGE, followed by transfer to membranes. The blots were incubated with the primary antibodies anti‐CD9, anti‐CD81, and anti‐HSP70, followed by incubation with rabbit anti‐Ig secondary antibodies (SBI System Biosciences). The specific bands were detected using the ECL chemiluminescent substrate (GE Healthcare Life Science) and then were visualized using the Amersham Imager 600 imaging system (GE Healthcare).
Ripa reagent
Manufactured by Merck Group
Sourced in United States, Canada
RIPA reagent is a lysis buffer used for the extraction and solubilization of proteins from cells and tissues. It contains a combination of detergents, salts, and other agents that help to disrupt cell membranes and release cellular proteins. The RIPA reagent is commonly used in biochemical and molecular biology applications, such as protein analysis, immunoprecipitation, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Protease inhibitor, by Roche (3 mentions)
β actin, by ABclonal (3 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Bca kit, by Merck Group (3 mentions)
Ab6721, by Abcam (3 mentions)
Ab125066, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Nr5a1, by Thermo Fisher Scientific (2 mentions)
Ab175186, by Abcam (2 mentions)
Protease inhibitor and phosphatase inhibitor, by Merck Group (2 mentions)
Ab8805, by Abcam (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
Ab193759, by Abcam (2 mentions)
Ab205719, by Abcam (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Ecl chemiluminescent substrate, by GE Healthcare (1 mentions)
26 protocols using ripa reagent
1
Extracellular Vesicle Protein Profiling
2
Western Blot Analysis of Protein Expression
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The cultured cells were lysed using the RIPA reagent (Millipore, USA) and the concentration of the proteins was quantified by the BCA protein assay kit (Bio-Rad, USA). The samples were subjected to 8% SDS-PAGE for electrophoresis and transferred onto the polyvinylidene fluoride membranes (Millipore). Then, the membrane was blocked with 5% skimmed milk diluted in Tris-buffered saline (TBS) containing 0.1% of Tween 20 (TBST buffer) for 1 hr at room temperature. The protein-specific primary antibodies of anti-VEGFA, anti-LRIG2, and anti-GAPDH (Abcam, USA) were added, then the protein was incubated at 4°C overnight. Subsequently, the membranes were rinsed by TBST buffer and probed with secondary horseradish peroxidase (HRP) conjugated antibodies for 1 hr at room temperature. Then, the proteins were determined by the enhanced chemiluminescence reagent (Millipore) in accordance with the manufacturer’s protocol and the bands were quantified using the ImageJ software.
3
Western Blotting and ELISA for Estradiol
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For western blotting, the protein was isolated from tissues or cells using RIPA reagent (Millipore, Bedford, MA, USA) supplemented with protease inhibitor (Roche, Mannheim, Germany), subjected to electrophoresis and membrane transfer, and then detected with antibodies of EEF2 (ABclonal Technology), NR5A1 (Invitrogen) and β-Actin (ABclonal Technology).
For ELISA, the concentration of estradiol in serum or cell culture medium was examined using the Mouse Estradiol ELISA Kit (Colorful Gene Biological Technology, Wuhan, China) according to the manufacturer’s protocol. Briefly, the samples were successively added to the micropores coated with mouse estradiol monoclonal antibody and then combined with horseradish peroxidase-labeled estradiol antibody to form antibody-antigen-enzyme labeled antibody complexes. After thorough washing, the substrate was added for color display. The concentration of estradiol in the sample was then calculated by measuring the absorbance at 450 nm using a microplate reader.
For ELISA, the concentration of estradiol in serum or cell culture medium was examined using the Mouse Estradiol ELISA Kit (Colorful Gene Biological Technology, Wuhan, China) according to the manufacturer’s protocol. Briefly, the samples were successively added to the micropores coated with mouse estradiol monoclonal antibody and then combined with horseradish peroxidase-labeled estradiol antibody to form antibody-antigen-enzyme labeled antibody complexes. After thorough washing, the substrate was added for color display. The concentration of estradiol in the sample was then calculated by measuring the absorbance at 450 nm using a microplate reader.
4
Western Blotting and ELISA for Estradiol
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For western blotting, the protein was isolated from tissues or cells using RIPA reagent (Millipore, Bedford, MA, USA) supplemented with protease inhibitor (Roche, Mannheim, Germany), subjected to electrophoresis and membrane transfer, and then detected with antibodies of EEF2 (ABclonal Technology), NR5A1 (Invitrogen) and β-Actin (ABclonal Technology).
For ELISA, the concentration of estradiol in serum or cell culture medium was examined using the Mouse Estradiol ELISA Kit (Colorful Gene Biological Technology, Wuhan, China) according to the manufacturer’s protocol. Briefly, the samples were successively added to the micropores coated with mouse estradiol monoclonal antibody and then combined with horseradish peroxidase-labeled estradiol antibody to form antibody-antigen-enzyme labeled antibody complexes. After thorough washing, the substrate was added for color display. The concentration of estradiol in the sample was then calculated by measuring the absorbance at 450 nm using a microplate reader.
For ELISA, the concentration of estradiol in serum or cell culture medium was examined using the Mouse Estradiol ELISA Kit (Colorful Gene Biological Technology, Wuhan, China) according to the manufacturer’s protocol. Briefly, the samples were successively added to the micropores coated with mouse estradiol monoclonal antibody and then combined with horseradish peroxidase-labeled estradiol antibody to form antibody-antigen-enzyme labeled antibody complexes. After thorough washing, the substrate was added for color display. The concentration of estradiol in the sample was then calculated by measuring the absorbance at 450 nm using a microplate reader.
5
Protein Isolation and Western Blot
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To prepare protein for western blot, the cultured cells were lysed with RIPA reagent (Millipore) supplied with protease inhibitor cocktail (Biomake). Then the same amount of total protein was subjected to SDS-PAGE separation and incubation with primary antibodies at room temperature for one hour, followed by secondary antibody incubation at room temperature for two hours. The anti-CIT antibody was purchased from Abcam (ab110897), an anti-CYPA antibody from Santa Cruz Biotechnology (sc-134310), an anti-GAPDH antibody from ProteinTech (60004-1-Ig), an anti-HIF1A antibody from ProteinTech (20960-1-AP).
6
Quantifying lncRNA and Protein Levels
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The relative expression levels of lncRNA-Gm2044 and mRNAs were detected by real-time Quantitative PCR (RT-qPCR ). The RNA of male germ cells was reverse transcribed to cDNA by the Evo M-MLV RT Kit (Accurate Biotechnology, Changsha, P. R. China) with RTase Reaction Buffer Mix, Evo M-MLV RTase Enzyme Mix, Oligo dT (18T) Primer and Random 6 mers Primer at 37°C 15 min, 85°C 5 s and 4°C hold. Afterwards, the cDNA was analyzed by RT-qPCR to study lncRNA-Gm2044 and mRNA expression using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) at 95°C 30 s, 40 cycles of 95°C 5 s and 60°C 30 s. The primers for lncRNA-Gm2044 and β-actin (internal reference) are listed in our previous research (Hu et al., 2018 (link), 2019 (link)), and other primers are shown in Table S3.
The expression level of the target protein was performed by western blotting. The protein was extracted from cells using the RIPA reagent (Millipore, Bedford, MA) with a protease inhibitor (Roche, Mannheim, Germany), separated with SDS-PAGE, transferred to nitrocellulose membranes, and treated with antibodies of GFP (Thermo Fisher Scientific, Rockford, IL), Flag (Affinity Biosciences, Cincinnati, OH), and β-actin (ABclonal Technology, Wuhan, China).
The expression level of the target protein was performed by western blotting. The protein was extracted from cells using the RIPA reagent (Millipore, Bedford, MA) with a protease inhibitor (Roche, Mannheim, Germany), separated with SDS-PAGE, transferred to nitrocellulose membranes, and treated with antibodies of GFP (Thermo Fisher Scientific, Rockford, IL), Flag (Affinity Biosciences, Cincinnati, OH), and β-actin (ABclonal Technology, Wuhan, China).
7
ESCC Protein Extraction and Western Blot
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Protein extraction was conducted through homogenizing ESCC cells in radioimmunoprecipitation assay (RIPA) reagent (Sigma-Aldrich, USA) supplemented with protease inhibitors. The homogenate was centrifugated at 12,000 rpm at 4°C for 20 min, and the supernatant was harvested. The concentration of proteins was assessed with bicinchoninic acid (BCA) protein assay kit (Pierce, USA). Afterwards, the proteins were loaded onto SDS polyacrylamide gel, separated through electrophoresis, and transferred onto nitrocellulose membranes via electroblotting. The membranes were blocked by 5% skim milk for 1 h at RT, followed by incubation with primary antibody of ACSL4 (1/10000; ab155282; Abcam), SLC7A11 (1/1000; ab175186), HO-1 (1/2000; ab52947), GPX4 (1/2000; ab41787), or GAPDH (1/10000; ab128915) diluted in 5% skim milk overnight at 4°C. After washing with PBS, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated anti-IgG antibody (1/500; ab7085) for 1 h at RT. Signal was visualized with enhanced chemiluminescence (ECL) and viewed utilizing FluorChem® M MultiFluor system (Cell Biosciences, USA).
8
Western Blot Protein Quantification Protocol
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Tissues and harvested cells were lysed and ultrasonicated in RIPA reagent containing protease inhibitor and phosphatase inhibitor (Sigma-Aldrich) on ice for 30 min. The supernatant was collected after centrifugation at 14,000 × g for 10 min at 4 °C. The protein concentration was measured by bicinchoninic acid (BCA) assay. Protein extracts were resolved on 10% sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gels (Wuhan Boster Biological Technology Ltd., Wuhan, China) prior to being transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking in 5% skim milk for 2 h, the membrane was incubated in primary antibody (Table S7 ) overnight at 4 °C and then incubated with secondary antibody: goat anti-rabbit IgG or goat anti-mouse IgG (Table S8 ) for 2 h at room temperature. Our bands were visualized with enhanced chemiluminescence kit (Thermo Scientific Fisher, Waltham, MA, USA) on a Tanon-5200 ECL imager (Tanon, Shanghai, China). The protein levels were quantified using Image J software. Target protein bands were normalized to the loading control GAPDH.
9
Protein Expression Analysis in CRC
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Total proteins were extracted from CRC tissue and cell samples by the RIPA reagent obtained from Sigma, USA, and separated using 10% SDS-PAGE. The isolated proteins were transferred into PVDF membranes followed by 5% nonfat milk incubation to minimize the noise. Next, the membranes were immersed in milk containing primary antibodies against SOCS3 (Rabbit, 1:2000, ab16030, Abcam, UK), CD133 (Rabbit, 1:1000, ab216323, Abcam), SOX2 (Mouse, 1:2000, ab171380, Abcam), OCT4 (Rabbit, 1:5000, ab109183, Abcam), AKT (Rabbit, 1:500, ab8805, Abcam), STAT3 (Mouse, 1:5000, ab119352, Abcam), p-AKT (Rabbit, 1:1000, ab38449, Abcam), p-STAT3 (Rabbit, 1:5000, ab76315, Abcam), and GAPDH (Rabbit, 1:3000, ab124905, Abcam), and incubated overnight. After excess primary antibodies were washed off by PBS, the membranes were probed by HRP-conjunct secondary antibodies, and the signals were visualized using the ECL reagent (Bio-Rad).
10
Western Blot Analysis of Migration and Apoptosis
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Cells were cultured with or without B1 for 48 h, and then both adherent and floating cells were harvested. After being washed with ice-cold PBS and lysed with RIPA reagent (Sigma-Aldrich) for 30 min at 4°C, the cells were centrifuged at 13,500 rpm for 15 min. Regarding western blotting, equal amounts of total protein were loaded onto glyceraldehyde 3-phosphate dehydrogenase (SDS-PAGE), and the separated proteins were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). Regarding the blocking of a non-specific binding, the PVDF membrane was soaked in PBS solution containing 5% nonfat milk at room temperature for 1 h. The membranes were probed for migration and apoptosis regulatory protein levels using specific primary antibodies, followed by peroxidase-conjugated appropriate secondary antibodies, which were visualized using enhanced chemiluminescence.
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