The collagen type І and β-actin antibodies were purchased from Thermo Scientific (MA1–26771). Protein was extracted from uterine tissue using RIPA lysis buffer which was obtained from Bio BASIC Inc. (Marhham, Ontario, Canada). The extracted protein was separated by SDS-PAGE on 4–20% polyacrylamide gradient gels. After incubation in 5% nonfat dry milk, Tris-HCL, and 0.1% Tween 20 for 1 h, collagen-I monoclonal antibody and β-actin antibody were added to one membrane containing specimen samples and incubated at 4 °C overnight. Appropriate secondary antibodies were incubated for 2 h at room temperature. After washing twice with 1× TBST, a densitometric analysis of the immunoblots was performed to quantify the amounts of collagen I and β-actin against the control sample by total protein normalization using Image analysis software on the Chemi Doc MP imaging system (Version 3) produced by Bio-Rad (Hercules, CA).
Ripa lysis buffer
Manufactured by Bio Basic
Sourced in Canada, United States
RIPA lysis buffer is a detergent-based buffer solution used for the extraction and solubilization of proteins from cells and tissues. It contains a combination of ionic and non-ionic detergents that help to disrupt cell membranes and release proteins. The buffer is suitable for a wide range of downstream applications, such as protein quantification, Western blotting, and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Bradford protein assay kit, by Bio Basic (2 mentions)
Protease inhibitor cocktail, by Bio Basic (2 mentions)
Pellet pestle, by Merck Group (2 mentions)
Odyssey licor fluorescent system, by LI COR (2 mentions)
Tgx 4 15 gradient gels, by Bio-Rad (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye 680cw goat anti rabbit igg, by LI COR (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phospho smad2 3, by Cell Signaling Technology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Jackson ImmunoResearch (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
18 protocols using ripa lysis buffer
1
Collagen I and β-Actin Quantification
2
SIRT1 and AMPK Modulation in Liver Cells
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Huh7 and HepG2 cells were harvested and lysed in RIPA lysis buffer supplemented with protease inhibitors (Bio Basic Inc.). A bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) was used to quantify protein levels in each sample, following which 30 µg protein/sample were separated via 10% SDS-PAGE prior to transfer onto PVDF membranes (Millipore Sigma). The blots were then blocked using either 5% BSA (Gibco; Thermo Fisher Scientific, Inc.) for AMKP and phosphorylated (p)-AMPK or non-fat milk in TBST (for other proteins), followed by overnight incubation with antibodies specific to SIRT1 (1:1,000; cat. no. 8469), AMPK (1:1,000; cat. no. 5831), p-AMPK (Threonine 172; 1:1,000; cat. no. 8208) and GAPDH (1:1,000; cat. no. 5174; all from Cell Signaling Technology, Inc.) at 4˚C. The blots were subsequently probed for 1 h at 4˚C with an appropriate horseradish peroxidase-linked rabbit (1:10,000; cat. no. 7074) and anti-mouse IgG (1:10,000; cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies followed by enhanced chemiluminescent substrate visualization (GE Healthcare). ImageJ Software version 1.46 (National Institutes of Health) was used for densitometric analyses.
3
Western Blot Analysis of STAT3 and Smad Signaling
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Kidney tissues were homogenized using RIPA lysis buffer (BioBasic) containing protease inhibitor cocktail (ThermoFisher, Waltham, MA, USA) and PhosSTOP™ phosphatase inhibitor (Roche, Basel, Switzerland). Homogenates were centrifuged at 20,000× g for 30 min to collect the supernatant and protein contents were measured by BCA assay (ThermoFisher, Waltham, MA, USA). Tris-glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted to separate the equal amounts of protein lysate. Then, proteins were electroblotted onto polyvinylidene difluoride membranes (Roche), blocked, and incubated with rabbit or mouse monoclonal antibodies against STAT3 (Proteintech, Rosemont, IL, USA), phospho-STAT3, Smad2/3, phospho-Smad2/3 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Abcam, Cambridge, UK) at 4 °C for 12 h, rinsed and incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at 4 °C for 1 h. The Western blot image was visualized by the HRP-substrate and UVP Imaging System, quantified using the VisionWorks software version 8.21, and normalized to GAPDH.
4
Anticancer Peptide Evaluation Using LPPC
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Lipo-PEI-PEG-complex (LPPC) was provided by the lab of Dr. Kuang-Wen Liao, National Yang Ming Chiao Tung University, Hsinchu, Taiwan. Anticancer peptides (ACP) were synthesized and purchased from AngeneBiotech (Taipei, Taiwan). Phenol, chloroform, RIPA lysis buffer and protease inhibitor were purchased from Bio Basic Inc. (Toronto, Canada). MTT, propidium iodide (PI), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Pierce™ ECL Western Blotting Substrate was purchased from Thermo (Thermo Fisher Scientific Inc., Waltham, MA, USA). Myco5A medium, M199 medium, EMEM medium, DMEM medium, RPMI-1640 medium, fetal bovine serum (FBS), PBS buffer solution, sodium pyruvate (100 mM), and 0.05% trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA).
5
Kidney Protein Extraction and Western Blot Analysis
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Kidney tissue protein extraction was carried out using RIPA lysis buffer (Bio Basic Inc., Canada), with added protease inhibitor cocktail. SDS-PAGE electrophoretic system via acrylamide Kit (TGX Stain-Free™ FastCast™, Bio-Rad Laboratories, USA) was used for the separation of equivalent volumes of the extracted proteins from all studied groups. The membranes were blocked with 5% non-fat dry milk, Tris-HCL, 0.1% Tween 20 for 1 h, and then incubated with the primary antibodies at 4 °C overnight. The utilized antibodies were p38 MAPK (Creative Biolabs, USA, Cat.# CBMAB-Z0329-LY), Nrf2 (Santa Cruz Biotechnology, USA, Cat.# (A-10): sc-365949), HO-1 (Santa Cruz Biotechnology, USA, Cat.# (A-3): sc-136960), Bax (Santa Cruz Biotechnology, USA, Cat.# (2D2): sc-20067), and Bcl-2 (Santa Cruz Biotechnology, USA, Cat.# (C-2): sc-7382). That was followed by incubation with the suitable secondary antibodies at room temperature for 2 h. Densitometric analysis of the immunoblots was carried out to estimate the amounts of p38 MAPK, Nrf2, HO-1, Bax, and Bcl-2 against β-actin using ChemiDocTM MP imaging system (Bio-Rad Laboratories, USA) with image analysis software (version 3).
6
Protein Analysis of Knee Joint Samples
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Knee joint samples were processed using RIPA lysis buffer (PL005; BIO BASIC INC., Canada), with supplementary protease and phosphatase inhibitors. Protein concentration was determined in the lysed samples using Bradford Protein Assay Kit (SK3041; BIO BASIC INC., Canada). Proteins were separated by gel electrophoresis (SDS-PAGE) using TGX Stain-Free™ FastCast™ Acrylamide Kit (Bio-Rad Laboratories, USA). After the transfer of proteins from the gel to PVDF membrane, the membrane was blocked by tris-buffered saline with Tween 20 (TBST) and 3% bovine serum albumin (BSA) at room temperature for 1 h. Overnight incubation with each primary antibody was carried out against the blotted target protein at 4 °C, followed by rinsing with TBST. Afterward, incubation with HRP-conjugated secondary antibody was carried out for 1 h at room temperature, followed by rinsing with TBST. Finally, detection of bands was performed via chemiluminescence technique and CCD camera-based imaging, followed by quantification using ImageJ software (USA).
7
Immunoblotting of Metabolic Proteins
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Cells were lysed in RIPA lysis buffer (Bio Basic Inc., Markham, ON, Canada). A total of 20 µg protein was separated by electrophoresis on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA). These membranes were then blocked with 3% skimmed milk and probed with primary antibodies (anti-beta actin, anti-PGC1 alpha and anti-UCP1 from Santa Cruz Biotechnology, Dallas, TX, USA). Membranes were washed with TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibodies (Santa Cruz Biotechnology, USA). Finally, signals were detected with an enhanced chemiluminescence system (ATTO, Amherst, New York, NY, USA) using an ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA).
8
Quantification of Collagen-I Levels
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The collagen type І antibody used was purchased from Thermo Scientific (MA1-26771). Protein from uterine tissue was extracted by RIPA lysis buffer which was provided by Bio Basic Inc. (Markham, Ontario L3R 8T4, Canada). Extracted protein was separated by SDS/PAGE on 4–20% polyacrylamide gradient gels. After incubation in 5% non-fat dry milk, Tris/HCl, 0.1% Tween 20 for 1 h; collagen-I monoclonal antibody was added to one of the membranes containing specimen samples and incubated at 4°C overnight. Appropriate secondary antibodies were incubated for 2 h at room temperature. After being washed twice with 1× TBST, densitometric analyses of the immunoblots were performed to quantitate the amount of collagen-I against control sample by total protein normalization using image analysis software on the ChemiDoc MP imaging system (version 3) produced by Bio–Rad (Hercules, CA).
9
Protein Extraction and Analysis for MDM2 and E-cadherin
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The proteins were separated from each sample by Ready Prep TM protein extraction kit (Bio Rad Inc, USA), by RIPA lysis buffer (Bio BASIC INC, Canada) with additional protease inhibitor and phosphatase inhibitor buffer. Then, the samples were centrifuged at ∼16,000×g for 30 minutes at 4°C to separate the supernatant and the amount of the sample was measured with the Bradford Assay Kit (Bio Basic Inc, Canada). After the separation of the antigen samples by sodium dodecyl sulfate-polyacrylamide, gel electrophoresis (SDS-PAGE) gel (Bio-Rad Inc, USA) to the immunoblotting membrane (PVDF) membrane blocked with 3% bovine serum albumin (BSA) using Bio-Rad Trans-Blot Turbo. The MDM2 and E-cadherin proteins were incubated with their diluted primary at 4°C overnight and washed with TBST and secondary antibodies conjugated with HRP enzymes. Then, chemiluminescent signals were measured and the bands were analyzed using ChemiDoc MP Imager against beta-actin [23 (link)].
10
Skeletal Muscle Protein Extraction and Western Blotting
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