Mouse brain homogenates were prepared as described in our earlier study56 (link). Briefly, mice brain tissues were snap frozen in liquid nitrogen when harvested and the wet weight of the tissues (in mg) was determined using an electronic balance. The entire left hemisphere of the brain was digested with ice-cold 1x RIPA lysis buffer (Cell Signaling Technology) containing detergents (1% Nonidet P40 and 1% sodium deoxycholate) together with the protease inhibitor cocktail tablet (Roche). Lysates were homogenized using a hand held motorized pestle (Sigma-Aldrich) on ice. Tissue lysates were collected for protein quantification using BCA analysis (ThermoFischer Scientific)
Ripa lysis buffer
Manufactured by Cell Signaling Technology
Sourced in United States, China
RIPA lysis buffer is a solution used for the extraction and solubilization of proteins from cells and tissues. It contains a combination of detergents, salts, and other components that help disrupt cell membranes and release proteins into solution, enabling their subsequent analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (42 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (35 mentions)
Bca kit, by Thermo Fisher Scientific (24 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (17 mentions)
Protease inhibitor cocktail, by Merck Group (15 mentions)
Nitrocellulose membrane, by Bio-Rad (15 mentions)
β actin, by Cell Signaling Technology (14 mentions)
Protease inhibitor cocktail, by Roche (13 mentions)
Gapdh, by Cell Signaling Technology (12 mentions)
β actin, by Merck Group (11 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (10 mentions)
Polyvinylidene difluoride membrane, by Merck Group (10 mentions)
Bca protein assay, by Thermo Fisher Scientific (9 mentions)
Chemidoc xrs system, by Bio-Rad (8 mentions)
Pvdf membrane, by Bio-Rad (8 mentions)
Chemidoc mp imaging system, by Bio-Rad (8 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (8 mentions)
Gapdh, by Abcam (8 mentions)
Bca protein assay kit, by Beyotime (8 mentions)
Protease inhibitor, by Roche (7 mentions)
382 protocols using ripa lysis buffer
1
Mouse Brain Homogenization and Protein Extraction
2
Western Blot Protocol for Protein Analysis
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Total cell lysates were prepared using 1xRIPA lysis buffer (Cell Signaling, Danvers, MA, USA), and protein concentrations were determined using the BCA assay kit (Thermo Scientific, Waltham, MA, USA). Protein samples (20–30 µg per lane for cell samples and 1–50 µg per lane for tissue samples) were separated on a 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in TBST buffer (20 mM Tris Base, 137 mM NaCl, and 0.1% Tween 20 (pH 7.4)) containing 5% nonfat milk. Membranes were then immunoblotted with the indicated primary antibodies against proteins of interest at 4 °C overnight at 1:1000 dilution (or 1:4000 for sEH antibody) followed by 1 h incubation with secondary antibodies conjugated with horseradish peroxidase at 1:4000 dilution. Bands of the protein of interest were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Pittsburgh, PA). Densitometry of bands was quantified using Image Studio software (LI-COR Biosciences, Lincoln, NE, USA) or ChemiDoc XRS+ system with Image Lab software (Bio-Rad, Hercules, CA, USA).
3
Porphyromonas gingivalis Induces LC3 Expression
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Primary GECs seeded at a density of 2 × 105 on six-well plates, were infected with P. gingivalis ATCC 33277 at an MOI of 100 for 0.5, 1, 3, 6, 12, and 24 hours. Cells were lysed with1X RIPA lysis buffer (Cell Signaling) with protease and phosphatase inhibitors: 1mM PMSF; 0.1mM TLCK; 1mM NaF; 2mM N-ethylmaleimide; 1 mM sodium orthovanadate; and 10 µg ml−1 aprotinin. Equal amounts of total protein were determined by colorimetric Bradford Assay (Bio-Rad) and loaded onto a 15% polyacrylamide gel. After gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane using wet-transfer system and the membrane was blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% nonfat dry milk.
Polyclonal rabbit anti-LC3 antibody was used at a dilution of 1:1000 (Novus Biologicals) and detected using anti-rabbit HRP-conjugated secondary antibody at 1:1000 (Cell Signaling). The same membrane was further stripped and probed with mouse anti-ß-tubulin antibody antibody 1:1000 (Invitrogen) for loading control, followed by anti-mouse HRP-conjugated secondary antibody 1:1000 (Cell Signaling). Protein bands were visualized using enhanced chemiluminescence (ECL, GE Healthcare) and band intensities were examined using NIH ImageJ.
Polyclonal rabbit anti-LC3 antibody was used at a dilution of 1:1000 (Novus Biologicals) and detected using anti-rabbit HRP-conjugated secondary antibody at 1:1000 (Cell Signaling). The same membrane was further stripped and probed with mouse anti-ß-tubulin antibody antibody 1:1000 (Invitrogen) for loading control, followed by anti-mouse HRP-conjugated secondary antibody 1:1000 (Cell Signaling). Protein bands were visualized using enhanced chemiluminescence (ECL, GE Healthcare) and band intensities were examined using NIH ImageJ.
4
Western Blot Protein Analysis
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Total cellular proteins were harvested by 1x RIPA lysis buffer (Cell Signaling Technology, MA, USA) containing phosphatase inhibitor and protease inhibitor. The proteins were loaded and separated by SDS‒PAGE, followed by transfer to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was probed with the primary antibodies overnight at 4 °C. The blots were then incubated with secondary antibodies after rinsing three times with PBST. The blots were detected using an ECL detection reagent (4 A Biotech Co., Ltd., Beijing, China) and visualized with a scanner. Then quantified the bands were performed with ImageJ software. Positive signals were detected when the relative intensity of target proteins or phosphorylated proteins was compared with that of GAPDH or total proteins.
5
Cardiomyocyte Protein Expression Analysis
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Cardiomyocytes were lysed with 1x RIPA lysis buffer (Cell Signalling Technology, USA). After centrifugation, the lysates were clarified, and the supernatants fractions were isolated. Protein concentrations in cells were defined by the BCA protein assay. Approximately 30–50 μg of protein was loaded and separated by SDS-PAGE gels and then transferred to a PVDF membrane. After blocking the membrane with 5% nonfat milk, the following primary antibodies were used for western blot: Bcl-2, Bax, Akt, GSK-3β, phospho-Akt (Ser473), phospho-GSK-3β (Ser9) (Cell Signalling Technology, USA), gp91phox, p47phox, and β-actin (Santa Cruz Biotechnology, USA). Then the membrane was probed with appropriate secondary antibodies. Finally, the blots were visualized using a chemiluminescence system (Pierce Biosciences, USA). Image analysis software (GeneTools from SynGene) was used to quantify the immunoblots.
6
Brain Protein Analysis via Western Blotting
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Mouse brain homogenates were prepared as described in our earlier study7 (link), with ice-cold 1x RIPA lysis buffer (Cell Signaling Technology) containing detergents (1% Nonidet P40 and 1% sodium deoxycholate) with the protease inhibitor cocktail tablet (Roche). Tissue lysates were collected for protein quantification using BCA analysis (ThermoFischer).
The brain lysates were run using a 10% Tris-glycine polyacrylamide gel or NuPAGE® Bis-Tris gels (Life Technologies), and transferred onto nitrocellulose membrane before probing with specific primary and secondary antibodies. The protein bands were visualized by chemiluminescence on the Image Station 4000R (Carestream Health Inc).
Densitometry analysis of the bands was performed by measuring the optical densities of the targeted protein bands relative to the β-actin level from the same brain sample. For protein phosphorylation, the optical densities of the phosphorylated protein bands were measured relative to the targeted total protein level from the same brain sample. The analysis was performed using the NIH Image J software.
The brain lysates were run using a 10% Tris-glycine polyacrylamide gel or NuPAGE® Bis-Tris gels (Life Technologies), and transferred onto nitrocellulose membrane before probing with specific primary and secondary antibodies. The protein bands were visualized by chemiluminescence on the Image Station 4000R (Carestream Health Inc).
Densitometry analysis of the bands was performed by measuring the optical densities of the targeted protein bands relative to the β-actin level from the same brain sample. For protein phosphorylation, the optical densities of the phosphorylated protein bands were measured relative to the targeted total protein level from the same brain sample. The analysis was performed using the NIH Image J software.
7
Protein Expression Analysis via Western Blot
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For the Western blot analyses, cells were seeded in 6-well plates and lysed in 1X RIPA lysis buffer (Cell Signaling, USA) containing 1 % 0.1 M PMSF and 1 % 100X protease inhibitor cocktail solution (Genedepot, USA). The total protein amounts were normalized using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, USA) and a microplate reader (Tecan Group ltd., Switzerland), equipped at Drug Development Research Core Center. 20 μg of proteins were loaded onto a 10–15 % acrylamide gel for SDS-PAGE and then transferred to a 0.2 μm PVDF membrane (Pall Life Science, USA). 5 % skim milk or 5 % BSA was used for membrane blocking, and then the membranes were incubated with primary antibodies at room temperature (RT) for 3 h or at 4 °C overnight. The blots were washed 3 times with tris-buffered saline-0.1 % Tween20 (1X TBST) and incubated with HRP-conjugated secondary antibodies (GeneTex, USA). The protein bands were detected using ECL solution reagent (GE Healthcare, USA) and LAS-3000 (Fuji Photo Film Co., ltd., Japan). Captured images were evaluated with Multi-Gauge software (Fuji Photo Film Co. ltd.). All indicated primary antibodies were purchased from Thermo Fisher, Cell Signaling, Santa Cruz, Novus, or MBL, and the details are listed in Table S1 .
8
FGFR2 and FGF7 Knockdown Assay
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Cells were grown overnight at 2 × 103 cells per well in a 96‐well plate or 1.25 × 105 cells per well in a 6‐well plate (Corning Cat#353046) without penicillin/streptomycin. Three independent siRNAs targeting FGFR2 (Qiagen Cat#SI02623047, SI04380649, and SI04948909, Hilden, Germany) or FGF7 (Qiagen Cat#SI03064663, SI03074925, and SI03110163) were transfected alongside an AllStars Negative (Qiagen Cat#1027281) and Hs cell death control (Qiagen Cat#1027299) at a final concentration of 25 nm using RNAimax (Thermo Scientific Cat#13778075) according to the manufacturer’s instructions. Cell viability was assessed after 144 h or were harvested after 72 h in 1xRIPA lysis buffer (Cell Signaling Technologies Cat# 9806S, Danvers, MA, USA) or Trizol (Thermo Scientific Cat#15596018) for protein and qRT‐PCR assessment, respectively. Assays were in biological triplicate.
9
Immunoprecipitation and Western Blotting of Cellular Proteins
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Cells were resuspended in 1xRIPA lysis buffer (Cell Signaling Technology) supplemented with protease and phosphatase inhibitors (Thermoscientific), and 1 mM PMSF (Sigma). Subsequently, sonication (Misonix) is used for lysing resuspended cells on ice. After protein concentrations were determined using Biorad protein assay and measuring absorbance at 595 nm on a Backman Coulter, DU 800 Series UV/Vis Spectrophotometer, proteins were separated by using Novex™ NuPAGE® Gel Electrophoresis Systems (Invitrogen), transferred to nitrocellulose membranes by iBlot® Gel Transfer Device (Invitrogen) and incubated with following primary antibodies: β-Actin, p53 and p63 (4A4) (Santa Cruz), STAT3, pSTAT3 (Tyr-705) and PARP (Cell Signaling Technology), p73 (NeoMarkers, cocktail Ab-4), PUMA and IkB-alpha (Abcam) and Noxa (ProSci incorporated). Immunoprecipitation was performed with Dynabeads® (Invitrogen). Cell lysates were incubated with Anti-HA-Tag rabbit polyclonal antibody (Sigma-Aldrich) for 24h. Subsequently, lysates were incubated with pre-cleared Dynabeads® for 4h. After washing steps, western blot was performed with anti-p63 (4A4) antibody (Santa Cruz).
10
Western Blot Analysis of TLR4 Signaling
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Cells were ruptured on ice with 1x RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA), containing protease inhibitor (Roche) and phosphatase inhibitor (Thermo Fisher Scientific Inc.). Cell lysates were clarified by centrifugation, and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in gels were transferred onto polyvinylidine (PVDF) membranes (Bio-Rad), and these were blocked with 5% BSA (Gibco) in PBS-T. Membranes were then incubated with antibodies against TRIF (Abcam), Myd88 (Cell Signaling Technology), TLR4 (Invitrogen), phospho-TRAM (MyBioSource, San Diego, CA, USA), TRAM (R&D Systems), phospho-TIRAP (Y86; Abcam), TIRAP (Abcam), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), phospho-p65 (Ser536; Cell Signaling Technology), and p65 (Enzo Life Sciences, Inc.) overnight at 4°C. After washing with PBS-T, membranes were stained with peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) or peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). Target proteins were then detected using the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore).
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