Proteins were extracted from testicular tissue specimens using radioimmunoprecipitation assay buffer (RIPA, Abcam, Germany) supplemented with protease and phosphatase inhibitors and centrifuged. After determining the concentration of proteins using BCA Protein Assay Kit (Abcam, USA), an equal amount of the extracted protein in each sample was separated using SDS-PAGE and transferred to the nitrocellulose membrane. Following blockage of membranes in 5% non‐fat dry milk for 2 h at room temperature, the membranes were incubated with primary antibodies against Stra8 (1:1000, Abcam, USA) and Gfra1 (1:1000, MyBioSource, Inc.) at 4 °C overnight. Then, the membranes were washed three times with Tris buffered saline with 0.1%Tween 20 (TBS-T, Abcam, Germany) and incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:2000, Abcam, USA) for 1 h at room temperature. The expression rate of Stra8 and GFRA1 proteins was assessed using enhanced chemiluminescence. Finally, the intensity of the bands was normalized to GAPDH and analyzed using the ImageJ software.
Radioimmunoprecipitation assay (ripa)
Manufactured by Abcam
Sourced in United Kingdom
RIPA is a lysis buffer used for the extraction and solubilization of proteins from cells and tissues. It contains a combination of detergents, salts, and buffers that help to disrupt cell membranes and release proteins into solution, while maintaining their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Trans blot turbo pvdf polyvinylidenfluorid membrane, by Bio-Rad (2 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Tbs t, by Abcam (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Protran, by Cytiva (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
10 protocols using radioimmunoprecipitation assay (ripa)
1
Western Blot Analysis of Stra8 and GFRA1
2
Protein Extraction and Western Blot Analysis
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Protein lysates were generated by treating cells with lysis buffer (1x RIPA (Abcam), 1x cOmplete mini protease inhibitor (Roche)), followed by incubation on ice and centrifugation (13523 RCF, 20 min at 4 °C). The supernatants were collected and either used directly or stored at −80 °C. Protein concentrations were measured with the Qubit™ Protein Assay Kit (Invitrogen) and diluted to a final concentration of 1.25 µg/µL. Denaturing SDS page with NuPAGE 4–12% Bis-Tris gels (Invitrogen) was performed at 150 V in MOPS buffer (Invitrogen), followed by protein transfer onto Amersham™ Protran™ 0.45 µm NC membranes (GE Healthcare Life science) at 100 V for 90 min in transfer buffer (25 mM Tris, 192 mM Glycine). Membranes were blocked with filtered 5% milk in TBS-T buffer (50 mM Tris, 150 mM NaCl, 1% Tween-20, pH 7.6) followed by primary antibody (see Supplementary Information) incubation at 4 °C overnight at. The membranes were washed in TBS-T prior HRP coupled secondary antibody incubation (1:10,000) at room temperature followed by further washing steps. For detection Amersham ECL Prime Western Blotting Detection Reagents (Cytiva) and RX X-ray film (Fuji) were used according to manufactures protocol.
3
Quantitative Analysis of NF-κB-p65 in Rat Brain
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Brain tissue isolated from rats, after rinsing with a cold PBS buffer, was crushed and homogenized in a microtubule with 1 mL RIPA (radioimmunoprecipitation assay) buffer (Abcam, Cambridge, UK) and in 1% protease inhibitor (Sigma) in a homogenizer device. After centrifugation at 13,000 rpm for 15 min, the supernatant was collected and the protein concentration was determined using a BCA kit (Thermo, Pierce, USA). 60μg total protein was electrophoresed on 10% polyacrylamide gel at a constant voltage of 80V for 35 min and then 120V for 45 min. The electrophoresed proteins were then transferred to polyvinylidene fluoride (PVDF) paper and incubated with NF-κB-p65 (Cell Signaling, USA) and beta-actin (β-actin) monoclonal antibodies at 4°C overnight. PBST solution containing 0.1% Tween-20 was used to wash the membrane and then HRP-Rabbit anti-rat secondary antibody (1:10,000) against the primary antibody was incubated on the shaker for 190 min and then washed again. Finally, by adding chemiluminescence ECL (GE Healthcare, Uppsala, Sweden) to the PVDF paper, the presence of NF-κB-p65 protein in the samples was investigated. In addition to the samples, the negative control sample (without primary antibodies) was also electrophoresed with the same concentration of proteins. Western blot test results were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).
4
Quantifying β-actin in Cell Lysates
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The concentration of β-actin in cell lysate samples was evaluated by means of an enzyme-linked immunosorbent assay (ELISA). HSC-3 cells (105 cells/well) were cultured into 24-well plates for overnight and treated by each transfection reagents for 48 h. Then, the cells were harvested using radio immunoprecipitation assay buffer (RIPA, Abcam, UK). Commercially available human ACTB/β-actin ELISA kit (LifeSpan BioSciences, Inc. Seattle, WA) was used to measure the levels of β-actin, according to the manufacturer’s instructions. The optical density was quantified using a multi-detection microplate reader, SpectraMax® M5 (Molecular Devices, Sunnyvale, CA), at 450 nm wavelength.
5
Modulation of HepG2 Lipid Metabolism
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Human hepatic cells (HepG2) were plated at a density of 106 cells per well on a six-well cell culture plates; were transfected with control, miRNA mimics or inhibitors (80 nM, Dharmacon), or siRNA (40 nM, Dharmacon) using Lipofectamine® RNAi MAX (Invitrogen); and subsequently stimulated with GW3965 (1 μM, Sigma-Aldrich) or simvastatin (5 μM, Sigma-Aldrich) for an additional 24 h. For cells treated with simvastatin, the medium was replaced with a cholesterol-depleted medium [DMEM containing 5% bovine lipoprotein-deficient serum (LPDS)], 24 h before simvastatin treatment. After treatment, cells were harvested and lysed with either TRIzol Reagent for RNA expression analysis or radioimmunoprecipitation assay buffer (RIPA, Abcam) for protein expression analysis.
6
Isolation of Endothelial Cell-Derived Extracellular Vesicles
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A modified differential centrifugation method was used to collect the bulk EC-EV containing large EV (microversicle) and small EV (exosomes) from cell culture supernatant of unstimulated (uEV), TNF-α stimulated (tEV), and cell-free medium (cEV). Briefly, collected supernatant from the same number of parent cells was first centrifuged at 300 g for 5 min at 4°C to eliminate cell debris. To remove remaining debris and apoptotic bodies, another centrifugation step was done on the supernatant passed through a 0.22-µm filter (VWR, Belgium) for 20 min at 2,000 g at 4°C (14 (link)). Afterward, to pellet the EC-EV, the supernatant was centrifuged at 110,000 g for 3 h at 4°C. All ultracentrifugation (UC) steps were performed using an L-90 Beckman centrifuge (Beckman Instruments, Inc., Fullerton, CA, USA) equipped with a Ti-70 rotor (Beckman Instruments) (15 (link)). Based on the downstream analysis, pellets were suspended in 1 ml of HEPES (Lonza), RIPA or extraction buffers (Abcam).
7
Protein Isolation and Western Blotting
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Protein isolation for western blotting16 was carried out by RIPA (radioimmunoprecipitation assay) lysis (Abcam) and the BCA (bicinchoninic acid) assay (Thermo scientific). After loading equal amounts of protein per lane (30 μg protein), 7%–20% PROTEAN‐TGX (Tris‐Glycine eXtended) gels (BIO RAD), were electrophoresed at 250 V for 30–45 min and transferred on to a Trans‐Blot Turbo PVDF (polyvinylidenfluorid) membrane (BIO RAD) using a Trans‐Blot Turbo system (BIO RAD). Thereafter, the membranes were blocked in EveryBlot Blocking Buffer (BIO RAD) according to the manufacturer's antibody specification protocol for 5 min and incubated overnight at 4°C with the respective primary antibody [P‐AMPKalpha Rabbit mAB (T172) #2535, P‐ACC (Ser79) Rabbit mAB #11818, P‐Raptor (Ser792) Rabbit mAB #2083, p53 Rabbit mAB #2527, mTOR Rabbit Ab #2972, Akt Rabbit Ab #9272, cyclin D1 Rabbit mAB #55506, beta‐actin Rabbit mAB #8457, α‐tubulin Rabbit mAb #2125, Bcl2 Rabbit mAb #4223 and BAX Rabbit AB #2772 (CST)] at 1:1000 dilution. The membranes were washed using PBST (phosphate buffered saline + tween 0.025%) and incubated with an Anti‐rabbit IgG HRP‐linked AB 7074; (1:2000 CST). ChemiDoc MP Imaging System (BIO RAD) was used for band detection. Western blots were done in triplicate and representative blots are depicted.
8
Notch Pathway Protein Detection by Western Blot
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Western blotting was applied to detect Notch signaling pathway-associated proteins, which included NICD, Hes1, Hes5 and Hey1. The cells were lysed with RIPA (Abcam) and the supernatant was collected by a centrifugation at 12,000 × g at 4°C for 15 min. A BCA assay was used to determine the protein concentration. Protein lysates (25 µg/lane) were separated by 12% SDS-PAGE. The PVDF membrane (Bio-Rad Laboratories, Inc.) was transferred by a Trans-Blot Transfer Slot (Bio-Rad Laboratories, Inc.) and blocked with 5% fat-free milk for 2 h at room temperature. The primary antibodies (anti-NICD; Abcam; cat. no. ab8925; 1:800; anti-Hes1; Abcam; cat. no. ab71559; 1:700; anti-Hes5; Abcam; cat. no. ab25374; 1:600; anti-Hey1; Abcam; cat. no. ab22614; 1:800) were added following the kit protocol and shaken at room temperature for 2 h and then incubated at 4°C for 12 h. The HRP-conjugated secondary antibodies (rabbit anti-human IgG; Abcam; cat. no. ab6759; 1:8,000; rabbit anti-goat IgG; Abcam; cat. no. ab6741; 1:10,000; goat anti-rabbit IgG; Abcam, ab6721; 1:8,000) were added and incubated at room temperature for 1 5 h. Chemiluminescence detection was carried out using ECL reagent (SignalFire, cat. no. 6883, Cell Signaling Technology, Inc.). Densitometry was performed using Quantity One software version 2.4 (Bio-Rad Laboratories, Inc.)
9
Protein Isolation and Quantification
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Protein isolation was performed using RIPA (Radioimmunoprecipitation assay) lysis (Abcam, Cambridge, UK). Subsequently, a BCA (Bicinchoninic acid) assay (Thermo scientific, Rockford, IL, USA) was used for protein quantification. After loading equal amounts of protein per lane (30 μg protein), 7 to 20% Protean-TGX (Tris-Glycine eXtended) gels (BIO RAD, Hercules, CA, USA) was electrophoresed at 250 V for 30 to 45 min and transferred to a TransBlot Turbo PVDF (Polyvinylidenfluorid) membrane (BIO RAD) using a TransBlot Turbo system (BIO RAD). Membranes were blocked in EveryBlock Blocking buffer (BIO RAD) according to the manufacturer’s antibody specification protocol for 5 min and incubated overnight at 4 °C with the primary antibodies (AKT rabbit Ab #9272, beta-Actin Rabbit mAB #8457, HSP 90 Rabbit AB #4874, STAT3 rabbit mAB #12640; Cell Signaling Technology, Leiden, The Netherlands) at 1:1000 dilution. Thereafter, the membranes were washed using PBST (phosphate buffered saline + tween 0.025%) and incubated with an Anti-rabbit IgG HRP-linked AB 7074; (1:2000; CST, Denver, MA, USA). Band detection was performed using the ChemiDoc MP imaging system (BIO RAD). Western blotting was performed without replicates. The comparative quantification of Western blot results was carried out using the BIO RAD Image lab software, Version 6.1 (BIO RAD).
10
ELISA Quantification of Liver Cytokines
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The enzyme-linked immunosorbent assay (ELISA) method was used to assess the Toll-like receptor 4 (TLR4) secretion (MyBioSource, California, USA), interleukin-1 beta (IL-1β ) secretion (Abcam Cambridge, UK), and tumor necrosis factor-alpha (TNFα) (Abcam, Cambridge, UK) production by the liver. The total proteins of the extracted livers were lysed by RIPA (Abcam, Cambridge, UK) and centrifuged at 15,000 g for 30 min. The ratio of 1:20 supernatants/dilutions were seeded into coated microplates with antibodies to induce enzyme-substrate reaction. Standard solutions were used for drawing the standard curves. The amounts of proteins were examined in supernatant fractions by ELISA kits. The rate of absorbance was measured in triplicate at 450 nm 17 (link) .
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