MCF-7 cells were grown in 6-well plates and treated with dichloromethane extracts of S. atropatana and DMSO, as a control for 24h. After treatment, cells were lysed in RIPA Extraction Buffer (Thermo Scientific, Canada) and equal amounts of proteins (100 μg) from cell lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS- PAGE). Subsequently, blotting onto polyvinyl-difluoride (PVDF) membrane (Roche Diagnostics GmbH, Germany) was performed in 150 mA for one hour. The membrane was subsequently blocked with 4% skim milk, washed and incubated with specific primary antibodies of a mouse anti-PARP monoclonal antibody and anti-β-actin as a normalizing control (Roche Diagnostics GmbH, Germany) for overnight. Then the membrane was probed with horseradish peroxidase-conjugated secondary antibody. Protein detection was carried out by exposing the membrane to ECL western blotting detection system (Amersham Phamacia Biotech Inc, USA).
Ripa extraction buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
RIPA extraction buffer is a detergent-based lysis buffer used for the extraction of proteins from cells and tissues. It is designed to solubilize a wide range of proteins while preserving their native conformation and biological activity. The buffer contains a balanced mixture of ionic and non-ionic detergents, as well as other components that aid in the lysis and extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Roche (1 mentions)
Anti β actin, by Roche (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Sod activity kit, by Enzo Life Sciences (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Gradient tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated antibody, by Cell Signaling Technology (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab14933, by Abcam (1 mentions)
Ab58632, by Abcam (1 mentions)
Ab95462, by Abcam (1 mentions)
9 protocols using ripa extraction buffer
1
Evaluating S. atropatana Extracts on MCF-7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
MCF-7 cells were grown in 6-well plates and treated with dichloromethane extracts of S. atropatana and DMSO, as a control for 24h. After treatment, cells were lysed in RIPA Extraction Buffer (Thermo Scientific, Canada) and equal amounts of proteins (100 μg) from cell lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS- PAGE). Subsequently, blotting onto polyvinyl-difluoride (PVDF) membrane (Roche Diagnostics GmbH, Germany) was performed in 150 mA for one hour. The membrane was subsequently blocked with 4% skim milk, washed and incubated with specific primary antibodies of a mouse anti-PARP monoclonal antibody and anti-β-actin as a normalizing control (Roche Diagnostics GmbH, Germany) for overnight. Then the membrane was probed with horseradish peroxidase-conjugated secondary antibody. Protein detection was carried out by exposing the membrane to ECL western blotting detection system (Amersham Phamacia Biotech Inc, USA).
+ Expand
MCF-7 cells were grown in 6-well plates and treated with dichloromethane extracts of S. atropatana and DMSO, as a control for 24h. After treatment, cells were lysed in RIPA Extraction Buffer (Thermo Scientific, Canada) and equal amounts of proteins (100 μg) from cell lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS- PAGE). Subsequently, blotting onto polyvinyl-difluoride (PVDF) membrane (Roche Diagnostics GmbH, Germany) was performed in 150 mA for one hour. The membrane was subsequently blocked with 4% skim milk, washed and incubated with specific primary antibodies of a mouse anti-PARP monoclonal antibody and anti-β-actin as a normalizing control (Roche Diagnostics GmbH, Germany) for overnight. Then the membrane was probed with horseradish peroxidase-conjugated secondary antibody. Protein detection was carried out by exposing the membrane to ECL western blotting detection system (Amersham Phamacia Biotech Inc, USA).
2
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confluent layers of HMVECs were treated according to specific experiment designs. At the conclusion of treatment, the cells were washed with PBS thrice and lysed with RIPA extraction buffer (Thermo Fisher, USA) containing a cocktail of protease/phosphatase inhibitors. Samples were subsequently handled in the immunoprecipitation assay buffer (20 mM HEPES buffer pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol), where relevant immunoprecipitant antibody was incubated with the sample for 90 min at suitable dilution, with gentle mixing. The protein A/G PLUS–agarose beads (Santa Cruz Biotechnology) were subsequently added. Samples were centrifuged and washed with immunoprecipitation assay buffer for a total of four times. The final spun-down pellet was resuspended in SDS loading buffer and proceeded to be subjected to immunoblotting/western blot assays according to the method described above.
3
Measurement of Superoxide Dismutase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein from healthy-MSCs or CKD-MSCs was extracted using a RIPA extraction buffer (Thermo Fisher Scientific). SOD activity was measured using a SOD activity kit (Enzo, Basel, Switzerland). Briefly, 40 µg protein was added to each well. A master mix (150 µL) containing WST-1 reagent and xanthine oxidase was also added to each well. In this colorimetric assay, superoxide ions are generated from the xanthine oxidase catalyzed conversion of xanthine and oxygen to uric acid and hydrogen peroxide (H2O2). The superoxide anion then converts WST-1 to the color WST-1 formazan product. Following the addition of the xanthine oxidase solution (25 µL/well), the absorbance was measured at 450 nm every minute for 15 min using a microplate reader (BMG Labtech, Ortenberg, Germany). SOD activity was calculated following the manufacturer’s instructions.
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue/cells were lysed in RIPA extraction buffer (ThermoFisher Scientific) supplemented with 1X Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Protein concentration was measured using Pierce BCA-kit (ThermoFisher Scientific) and protein samples were separated by SDS-PAGE on a 4–20% TRIS-glycine gradient gel (ThermoFisher Scientific). Afterwards, the proteins were blotted onto a polyvinylidene fluoride membrane using iBlot system (Invitrogen). Target proteins were detected using specific antibodies and visualized with respective horseradish peroxidase (HRP) conjugated antibodies (Cell signaling, Danvers, MA, USA) and Clarity Western ECL substrate (Biorad, Hercules, CA, USA). Chemiluminescence was recorded by ChemiDoc Touch Imaging system (Biorad). After stripping the membranes using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) for 20 min, membranes were reprobed with β-Actin antibody (Cell signaling) and further processed as described above. Densitometric analysis of protein bands was performed using Imagelab Software (version 5.2). Western blot images were cropped for easier presentation. Originals are visualized in the supplementary material.
5
Western Blotting Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the primary antibodies applied are shown in Table S2 . There were four main steps in Western blotting. First, protein extraction was performed. Cells or tissues were lysed with RIPA extraction buffer (Thermo, 89900) containing protease and phosphatase inhibitor cocktails, and the supernatant solution was quantified after centrifugation. Second, SDS‒PAGE separated proteins through electrophoresis, and then the protein was transferred to PVDF membranes (Millipore, IPVH00010, ISEQ00010). Third, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) and incubated with antibodies according to the instructions. Finally, the bands were tested and analysed by ImageJ.
6
Immunoblotting Quantification of RAP1B Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from cell cultures using RIPA extraction buffer (Thermo Scientific, Carlsbad, CA, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Carlsbad, CA, USA). The proteins were separated through SDS-PAGE (12.5%) and subsequently transferred to nitrocellulose membranes, which were blocked with 5% skimmed milk and incubated with the primary anti-RAP1B antibody (CST, Danvers, MA, USA) at a 1:1000 dilution overnight at 4 °C. After washing with TBS-T, the membranes were incubated with anti-Rabbit secondary antibody coupled to HRP (Invitrogen, Carlsbad, CA, USA) at a 1:2000 dilution. An antibody against GAPDH was used as an endogenous control. The membranes were reused and incubated with primary anti-GAPDH antibody at a 1:2000 dilution (Invitrogen, Carlsbad, CA, USA) overnight at 4 °C. Pierce ECL Western Blotting Substrate (Thermo Scientific, Carlsbad, CA, USA) was used for development.
7
Osteoblast Differentiation Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary cells were extracted from the flat bone of P3 Ddr1f/f-4OHT and OKOΔDdr1 mice and MC3T3-E1 cells were extracted with RIPA extraction buffer (89900, Thermo Fisher Scientific Inc., Waltham, MA, USA.) containing 1% proteinase inhibitor (78429, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% phosphatase inhibitor (78426, Thermo Fisher Scientific Inc., Waltham, MA, USA). Western blot was performed with primary antibodies against DDR1 (ab22719, Abcam, Cambridge, MA, USA), collagen type I (ab34710, Abcam), collagen type X (ab58632, Abcam), GAPDH (MA5-15738, Thermo Fisher Scientific Inc.), ALP (ab95462, Abcam, Cambridge, MA, USA), Runx2 (ab23981, Abcam), BMP2 (ab14933, Abcam), Col-I (ab34710, Abcam), Osteocalcin (GTX13418, Gene Tex Inc., Irvine, CA, USA), phosphorylated 44/42 ERK1/2 (D11A8, Cell Signaling Inc., Danvers, MA, USA), ERK1/2 (137F5, Cell Signaling Inc.), phosphorylated p38 (Thr180/Tyr182) (ARG51580; Arigo biolaboratories, Hsinchu, TW), p38 (ARG55258; Arigo biolaboratories) and mouse and rabbit secondary antibodies (615005214 and 611005215; Jackson immune research; West Grove, PA, USA). Protein bands were detected by enhanced chemiluminescence analysis (ECL system; GE Healthcare, Piscataway, NJ, USA).
8
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from cells by using RIPA extraction buffer (Thermo Scientific) on ice for 30 min according to the manufacturer's instructions. The obtained supernatant was separated with a 10% SDS-PAGE gel and transferred to PVDF membranes. Then, the membrane was blocked with 15% milk for 1 h and incubated with primary antibodies against Bax (Cell Signaling Tech., 1 : 1000), caspase 3 (Cell Signaling Tech., 1 : 1000), and beta-actin (Protein Tech., 1 : 1000). After incubation with secondary antibody, protein signals were detected with a chemiluminescence kit (Thermo Fisher Scientific, Inc.). Pictures were captured with an automatic image scanning system (Gene, Co.).
9
Stretching-Induced Fibrinogen Binding
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!