Hepatic tissues were prepared for protein extraction. Total protein was acquired by using RIPA buffer (NO: G2002, Servicebio, China) with PMSF (G2008, Servicebio) for 30 min followed by centrifugation at 12000 rpm for 10 min at 4°C. Next, the nuclear protein was obtained by using a Nuclear Protein Extraction kit (G2007, Servicebio), the concentrations were determined by using BCA protein assay (G2026, Servicebio), the total protein was subjected to SDS-PAGE gel electrophoresis (G2003, Servicebio) and electroblotted onto PVDF membranes (ISEQ00010, Millipore, USA), and the membranes were blocked for 1 h in 5% BSA (G5002, Servicebio) and subsequently incubated with the following primary antibodies at 4°C overnight. Primary antibodies were ACTIN (mouse monoclonal antibody 1:1000, GB12001, Servicebio), GLUT4 (rabbit monoclonal antibody 1:1000, bs-0384, BIOSS, China), and AMPK (rabbit monoclonal antibody 1:1000, GB11627, Servicebio), and then, the membrane was incubated with the secondary antibody 1:3000 and the conjugates were visualized with an ECL system (G2014, Servicebio). Finally, the odyssey imaging system was used to analyze protein expression.
Ripa buffer
Manufactured by Wuhan Servicebio Technology
Sourced in China
RIPA buffer is a commonly used lysis buffer for extracting proteins from cells and tissues. It is a detergent-based buffer that helps to solubilize cellular components and release proteins. The buffer contains a mixture of ionic and non-ionic detergents, as well as other components that help to maintain protein integrity during the extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Wuhan Servicebio Technology (9 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Phosphatase inhibitor, by Wuhan Servicebio Technology (6 mentions)
Bca protein assay kit, by Wuhan Servicebio Technology (6 mentions)
Bca kit, by Wuhan Servicebio Technology (6 mentions)
Protease inhibitor cocktail, by Wuhan Servicebio Technology (5 mentions)
Pvdf membrane, by Wuhan Servicebio Technology (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ecl plus reagents, by Wuhan Servicebio Technology (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Anti p62, by Abcam (3 mentions)
Westernbright ecl kit, by Advansta (3 mentions)
Protease inhibitor, by Wuhan Servicebio Technology (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Gapdh, by Proteintech (2 mentions)
β actin, by Wuhan Servicebio Technology (2 mentions)
Anti occludin, by Abcam (2 mentions)
Anti bax, by Abcam (2 mentions)
Anti caspase 3, by Abcam (2 mentions)
85 protocols using ripa buffer
1
Hepatic Protein Extraction and Analysis
2
Western Blotting of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the western blotting of the total lysates, we lysed cells in RIPA buffer (Servicebio) supplemented with phosphatase and protease inhibitors (Servicebio), which in turn subjected the lysates to protein quantification (Beyotime). The lysates were immunoblotted, electrophoresed on SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with various antibodies. Chemiluminescent signals were detected by ChemiScope 6300 Touch (CLINX). Protein quantification was performed by Image J (NIH).
3
Kidney Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from kidney tissue using RIPA buffer (Servicebio, China) and detected with a BCA protein assay kit (Nanjing Jian Cheng Co., China) to determine the concentration of total protein. Protein was separated by SDS-PAGE, electrophoretically transferred to PVDF membranes, and then blocked in 5% nonfat milk. The protein bands were incubated at 4°C overnight with primary antibodies, including Bax, Bcl-2, caspase 3, and Cyto-c, followed by incubation with the secondary antibody for 2 hours at room temperature. Protein expression was detected using the Quantity One analysis system (BioRad, USA) and analyzed with ImageJ. β-Actin was used as an internal reference.
4
Quantification of Hypoxia-Inducible Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from GCs were homogenized using RIPA buffer (Servicebio, G2002, Wuhan, China). Protein concentrations were determined using the BCA Protein Assay Kit (Solarbio, PC0020, Wuhan, China). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore, IEVH00005, Burlington, MA, USA), and then incubated with antibodies (HIF1A, Novus, NB100, Littleton, CO, USA; GAPDH, Proteintech, 60004-1-Ig, Wuhan, China) overnight at 4°C and then with HRP-conjugated secondary antibody for 1 h at room temperature. Pictures were captured by an imaging system (UVP).
5
Western Blot Analysis of Brain Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were decapitated at the time of each experimental procedure, and the right cerebral hemisphere was separated and homogenized in RIPA buffer (Servicebio, Wuhan, China). Western blotting was performed as previously described [20 (link)]. The primary antibodies included anti-MST4 (1 : 1000, Proteintech), anti-AKT (1 : 1000, Cell Signaling Technology), anti-pAKT (1 : 1000, Cell Signaling Technology), anti-LC3A/B (1 : 1000, Cell Signaling Technology), and β-actin (1 : 1000, Cell Signaling Technology).
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was extracted from liver and colon tissues using RIPA buffer (Servicebio) and concentrations were detected using a BCA protein analysis kit (Servicebio). Proteins from the different groups of samples were separated by electrophoresis using SDS-PAGE gels transferred onto PVDF microporous membranes. The transferred membranes were placed in an incubation bath with TBST and washed once quickly, then skimmed milk was added and placed on a decolorization shaker and closed for 30 min at room temperature; primary antibodies were added and incubated at 4°C for 24 h with slow shaking; after washing the membranes three times with TBST, the membranes were incubated with secondary antibodies for 2 h at room temperature and again with TBST for rapid elution; finally the blots were visualized using ECL reagents Visualization. Protein band densities were analyzed by ImageJ software.
7
Western Blot Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissues were lysed with RIPA buffer (Servicebio) supplemented with protease and phosphatase inhibitor cocktail (PMSF, Servicebio; NaF, and NaV2O4). Total lysates were resolved on 10% SDS-PAGE gels (Epizyme) and transferred onto PVDF membranes at 0.2A (1.5 h, 4 °C). Then, the membranes were blocked with 5% non-fat milk at room temperature for 1 hour. Afterwards, the membranes were probed with antibodies against P2Y2, P2Y6, GSK-3β, β-catenin, Akt, p-Akt (pan) (all CST), N-cadherin (Proteintech), E-cadherin (Sevicebio), and β-actin (Servicebio) diluted in PBST overnight at 4 °C. Thereafter, appropriate secondary antibodies (Proteintech) were incubated for 1 hour at room temperature before washing three times and detecting with an ECL reagent. The western blot images were acquired using the ChemiDocTMXRS+ system (BioRad).
8
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared using the radio-immunoprecipitation assay (RIPA) buffer (Servicebio, Wuhan, China) containing a protease inhibitor cocktail (Roche, IN, USA). Western blot analysis of target proteins was conducted as described using the indicated primary antibodies, followed by probing to the corresponding HRP-conjugated secondary antibody. The reactive bands were visualized using ECL plus reagents (Servicebio, Wuhan, China). GAPDH was used as an internal control to analyze the results. The detailed information about the antibodies was as following:
Anti-BCKDE1a (#90198), anti-BCAT2 (#79764), anti-NOTCH4 (#2423), anti-c-myc (#14819) and anti-fatty acid synthase (FAS, #3180) antibodies were obtained from the Cell Signaling Technology (Danvers, MA, USA). Anti-CPT1a antibody (#15184-1) was purchased from Proteintech Group (Wuhan, China). Antibody against GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-BCKDE1a (#90198), anti-BCAT2 (#79764), anti-NOTCH4 (#2423), anti-c-myc (#14819) and anti-fatty acid synthase (FAS, #3180) antibodies were obtained from the Cell Signaling Technology (Danvers, MA, USA). Anti-CPT1a antibody (#15184-1) was purchased from Proteintech Group (Wuhan, China). Antibody against GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
9
Comprehensive Lung Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scientific, Wuhan, China), followed by electrophoresed through 8–12% sodium dodecyl sulfate–polyacrylamide gel and then transferred to 0.45 μm PVDF membrane (Merck Millipore, USA). A total of 30 ug of protein was used for western blot experiments. After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1 h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).
10
Western Blot Protein Detection Protocol
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!