VICs were washed two times in PBS, solubilised and homogenised in RIPA buffer solution (Sigma) supplemented with protease inhibitor cocktail 1X (Roche). Cells were scraped with a rubber policeman; the lysate was transferred into a 1.5ml microtube and vortexed. Proteins were quantified with a Pierce BCA protein assay (Thermo Scientific) after a 10,000g centrifugation for 10 minutes at 4°C. Total protein homogenates (7.5 μg) were denatured and separated on 10% Bis-Tris gels (Invitrogen). Electrophoretically resolved bands were then transferred on nitrocellulose membranes (Hybond C, Amersham). Membranes were blocked for 1 hour in Phosphate-Buffered Saline (PBS) containing 0.1% Tween-20 (PBS-T) and 5% (w/v) non-fat powdered milk. Then, they were incubated for 1 hour with primary antibodies (Table 1 ) in PBS-T containing 5% (w/v) non-fat powdered milk. Membranes were then washed three times in PBS-T and incubated with corresponding horseradish peroxidase conjugate secondary antibody (Table 1 ) for 1 h at room temperature in PBS-T. Membranes were washed five times in PBS-T. Visualization of the protein bands was accomplished using enhanced chemiluminescence (ECL) substrate (Amersham) and positivity was captured on Hyperfilm (Amersham). Films were scanned and bands were quantitated using the QuantityOne program (Biorad). Levels of expression were normalised to GAPDH.
Ripa buffer solution
Manufactured by Merck Group
Sourced in United States
RIPA buffer solution is a commonly used lysis buffer in biochemistry and cell biology laboratories. It is designed to extract proteins from cell and tissue samples for further analysis. The buffer contains a combination of detergents, salts, and buffers that help solubilize and denature proteins, while maintaining their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Phosstop, by Roche (2 mentions)
Hif1a, by Abcam (2 mentions)
P akt, by Abcam (2 mentions)
Chsy1, by Abcam (2 mentions)
Peroxidase bound secondary antibodies, by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Pd l1, by Abcam (2 mentions)
P pi3k, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hyperfilm, by Cytiva (1 mentions)
Protease inhibitor cocktail 1x, by Roche (1 mentions)
Hybond c, by Cytiva (1 mentions)
Ecl substrate, by Cytiva (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Quantity one program, by Bio-Rad (1 mentions)
Trypan blue stain, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
13 protocols using ripa buffer solution
1
Western Blot Analysis of Protein Expression
2
Renal Adenocarcinoma Cell Culture Protocol
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Mouse renal adenocarcinoma-derived Renca cells (ATCC CRL-2947) and human renal adenocarcinoma-derived 786-O cells (300107, CLS Cell Lines Service, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F-12 (Gibco, 31331028), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5% CO2. For experiments 5 × 106 cells were plated per 15 cm dish (Greiner Cellstar, 639160). Cell viability was tested with Trypan Blue Stain (T10282 Invitrogen) in TC20 Automated Cell Counter (145-0101 Biorad) and was in the range of 95–97%. The whole-cell lysates were prepared by adding 2 ml of lysis buffer (RIPA buffer solution (R0278 Sigma-Aldrich) with proteinase (P5726 Sigma) and phosphatase (50892791001 Roche) inhibitors) per 15 cm dish. After 3 min. incubation at RT, cell lysates were transferred to Eppendorf tubes and further incubated on shacking platform for 30 min at + 4 °C. The proteins were pelleted by centrifugation at 15,000g for 20 min.
3
Protein Analysis in Cell Lysates
4
Quantifying BDNF Expression in Rat Tissues
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The soleus muscle and brain of rats were excised quickly, freeze-clamped in
liquid nitrogen, and stored at −80°C until use. For western blot analysis,
frozen tissue samples were homogenized in 10 volumes (wt/vol) of RIPA buffer
solution (Sigma-Aldrich, St. Louis, MO, USA). Proteins were quantified using a
BCA protein assay kit (Pierce, Rockford, IL, USA). Proteins were denatured by
heating for 5 min at 95°C in 0.5 M Tris-HCl buffer (pH 6.8) containing 10% SDS
and 10% ammonium persulfate, separated by electrophoresis on 17.5% or 10%
SDS-polyacrylamide gels, depending on protein size, and transferred to a
nitrocellulose membrane in 25 mM Tris buffer containing 15% methanol, 1% SDS,
and 192 mM glycine. After blocking for 1 hour with 5% skim milk in Tris-buffered
saline-Tween (TBS-T) (pH 7.6), the membrane was incubated with anti-BDNF (1:500;
rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at
4°C. After washing with TBS-T, the membrane was incubated with a secondary goat
anti-rabbit IgG conjugated with horseradish peroxidase (1:2,000; Santa Cruz
Biotechnology) for 2 hours at room temperature. The membrane was then developed
using an enhanced chemiluminescence solution (Pierce). The band densities were
measured using ImageJ software (National Institutes of Health, Bethesda, MD,
USA) and normalized to the density of β-actin.
liquid nitrogen, and stored at −80°C until use. For western blot analysis,
frozen tissue samples were homogenized in 10 volumes (wt/vol) of RIPA buffer
solution (Sigma-Aldrich, St. Louis, MO, USA). Proteins were quantified using a
BCA protein assay kit (Pierce, Rockford, IL, USA). Proteins were denatured by
heating for 5 min at 95°C in 0.5 M Tris-HCl buffer (pH 6.8) containing 10% SDS
and 10% ammonium persulfate, separated by electrophoresis on 17.5% or 10%
SDS-polyacrylamide gels, depending on protein size, and transferred to a
nitrocellulose membrane in 25 mM Tris buffer containing 15% methanol, 1% SDS,
and 192 mM glycine. After blocking for 1 hour with 5% skim milk in Tris-buffered
saline-Tween (TBS-T) (pH 7.6), the membrane was incubated with anti-BDNF (1:500;
rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at
4°C. After washing with TBS-T, the membrane was incubated with a secondary goat
anti-rabbit IgG conjugated with horseradish peroxidase (1:2,000; Santa Cruz
Biotechnology) for 2 hours at room temperature. The membrane was then developed
using an enhanced chemiluminescence solution (Pierce). The band densities were
measured using ImageJ software (National Institutes of Health, Bethesda, MD,
USA) and normalized to the density of β-actin.
5
Western Blot Analysis of POLD1 Protein
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Pelleted PBMCs or cultured cells were lysed with RIPA buffer solution (Sigma-Aldrich, MA, United States) supplemented with cOmplete Protease Inhibitor Cocktail and PhosSTOP (Roche, Basel, Switzerland).
Protein extracts were run in NuPAGE™ gels according to the manufacturer’s protocol (ThermoFisher, Waltham, MA) and transferred into PVDF membranes (Millipore, Bedford, MA). Blots were probed with anti-POLD1 (EPR15118, #ab186407, Abcam) or anti-GAPDH (clone 14C10, #2118, Cell Signaling) primary antibodies diluted 1:5000, followed by the incubation with the fluorescent Dylight 800 anti-rabbit secondary antibody (SA5-10036). Protein detection was carried out using Odyssey Imaging System (LI-COR, Lincoln, NE).
Protein extracts were run in NuPAGE™ gels according to the manufacturer’s protocol (ThermoFisher, Waltham, MA) and transferred into PVDF membranes (Millipore, Bedford, MA). Blots were probed with anti-POLD1 (EPR15118, #ab186407, Abcam) or anti-GAPDH (clone 14C10, #2118, Cell Signaling) primary antibodies diluted 1:5000, followed by the incubation with the fluorescent Dylight 800 anti-rabbit secondary antibody (SA5-10036). Protein detection was carried out using Odyssey Imaging System (LI-COR, Lincoln, NE).
6
Western Blot Protein Extraction and Analysis
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Total protein extracts, without prior pepsin digestion, were prepared using RIPA buffer solution (Sigma‐Aldrich) supplemented with protease and phosphatase inhibitor cocktail (1% [v/v], ThermoFisher). Total protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific BCA Protein Assay Kit). 4%–12% Bis‐Tris polyacrylamide gels (Invitrogen, NP0326BOX) were used for loading protein samples and subjected to electrophoresis (200 V, 50 min). Then semi‐dry transfer or wet transfer was used for transferring the protein blot from the gel to the membrane. Polyvinylidene fluoride membranes with low fluorescence background (Millipore, Billerica, MA, USA) were used in a wet transfer manner, and iBlot‐2 Transfer Stacks were used in a semi‐dry manner by iBlot 2 Dry Blotting System (Invitrogen). Membranes were blocked in 3% milk in Tris‐buffered saline with Tween‐20 (TBS‐T, 0.1% Tween‐20) for 1 h, and 1% milk/TBS‐T was used to incubate membranes overnight at 4°C with primary antibodies. Horseradish peroxidase (HRP)‐conjugated secondary antibodies (Thermo Scientific) were used to perform immunodetection followed by Super Signal West Dura Extended Duration Substrate (Thermo Scientific Pierce Protein Biology), and then imaged with ChemiDoc Touch Imaging system (Bio‐Rad, Hercules, CA). Antibody information is provided in Table S2 .
7
Quantifying Hippocampal AChE Activity
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Twenty-four hours following the fear conditioning test, mice were sacrificed for the collection of hippocampus and whole cerebellum. Hippocampi were further dissected into dorsal and ventral sections (1:1 ratio). All tissue was flash frozen on dry ice and stored at −80°C until further processing. Brain tissue from 5 to 6 mice was randomly selected from each strain. Tissue was homogenized in 1X RIPA buffer solution (R0278, Sigma Life Sciences, St. Louis, MO, USA) with HALT Protease and Phosphatase Inhibitor Cocktail at a ratio of 100:1 (78445, Fisher Scientific, Pittsburgh, PA, USA). Homogenates were spun down at 14,000 g at 4°C for 30 min. Total protein concentration of the supernatant was determined by DC Protein Assay (500-0112; Bio-Rad, Hercules, CA, USA). Each supernatant sample was further diluted in RIPA buffer to a standard 30 μg total protein. AChE activity in diluted samples was measured using the Abcam Acetylcholinesterase Assay Kit (ab138871, Abcam, Cambridge, MA, USA) per manufacturer instructions. Inter- and intra-assay duplicate CV% were all <15%.
8
Western Blot Analysis of Protein Signaling
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Proteins were extracted from cells with RIPA buffer solution (Sigma-Aldrich, USA), dissolved on SDS–polyacrylamide gel, and then transferred to PVDF membrane. Primary antibodies CHSY1, P-PI3K, PI3K, AKT, PD-L1, P-AKT and HIF1A were used (Abcam, UK). Using peroxidase-bound secondary antibodies (Sigma-Aldrich, USA), chemiluminescence (ECL; Thermo Fisher, USA).
9
Cytokine and Chemokine Quantification in Lung Homogenate
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Lung homogenate was added to the RIPA buffer solution (Sigma-Aldrich, cat. R0278) and centrifuged at 10,000 g at 4 °C for 10 min. The supernatant was collected. The ELISA method was performed to detect the concentration of cytokines and chemokines using kits from R&D Systems (DuoSet), according to the manufacturer’s instructions. The following targets were evaluated: TNF-α, IL-6, IL-10, CXCL1, CCL2, and CCL4.
10
Western Blot Analysis of Macrophage Signaling
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Macrophages were washed three times with ice‐cold PBS and lysed for 20 min on ice in RIPA buffer solution (Sigma‐Aldrich) with protease and phosphatase inhibitor cocktails (Sigma‐Aldrich). Equal amounts (20 mg) of cell lysates were resolved using 8%‐15% polyacrylamide gels transferred to PVDF membrane (Bio‐Rad, USA). Membranes were blocked in 5% non‐fat dry milk in PBS‐T and incubated overnight with their respective primary antibodies at 4°C. These respective primary antibodies' list are as follows: Phospho‐NF‐κB p65 (Ser536) (Clone: 93H1; CST, USA), NF‐κB p65 (Clone: D14E12; CST), Phospho‐p38 MAPK (Thr180/Tyr182) (Clone: D3F9; CST), p38 MAPK (Clone: D13E1; CST), Phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Clone: D13.14.4E; CST), p44/42 MAPK (Erk1/2) (Clone: 137F5; CST), Phospho‐JNK (Thr183/Tyr185) (Clone: G9; CST), SAPK/JNK (CST), SGPL1 antibody (No. ABIN207537, 4A Biotech Co., Ltd., China) and GAPDH (Clone: D16H11; CST). The membranes were incubated at room temperature for 1 h with appropriate HRP‐conjugated secondary antibodies and were visualized with Plus‐ECL (PerkinElmer, CA) according to the manufacturer's protocol.
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