Total proteins were extracted from cells in RIPA Lysis Buffer (Vazyme, Piscataway, NJ, USA) containing protease inhibitors. A total of 40 μg of protein from each sample was denatured, fractionated by 10% SDS-PAGE, and transferred to a PVDF membrane (Immobilon®-P Transfer Membrane, Millipore, Milan, Italy). After blocking nonspecific antigens with 5% skim milk solution, blots were incubated overnight at 4 °C with primary rabbit monoclonal antibodies against IL8 (1:1,000 working dilution, DF6998, Affinity Biosciences, Cincinnati, USA), CD274 (1:1,000 working dilution, DF6526, Affinity Biosciences, Cincinnati, USA), or β-actin (1:1,000 working dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 5% skim milk 0.05% TBS-Tween 20 buffer. Antibody binding to the membrane was detected with a secondary antibody (goat anti-rabbit IgG 1:5,000, ZSGB Biosciences, Beijing, China) conjugated to horseradish peroxidase and visualized by enzyme-linked chemiluminescence (EasySee® Western Blot Kit, TransGen Biotechnology, Beijing, China) with the Scientific MYECL Imager (Thermo, Boston, MA, USA). Densitometric analysis performed with ImageJ software was used to normalize the signals of IL8 and CD274. The intensity of the two bands was normalized against the signal of β-actin.
Ripa lysis buffer
Manufactured by Vazyme
Sourced in China
RIPA lysis buffer is a commonly used detergent-based buffer solution designed for the extraction and solubilization of proteins from cells and tissues. It contains a combination of ionic and nonionic detergents that help to disrupt cell membranes and release intracellular proteins. The buffer also contains other components that help to maintain the integrity and stability of the extracted proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Protease inhibitor, by Vazyme (4 mentions)
Anti c ebpα, by ZenBio (4 mentions)
Anti foxo1, by ZenBio (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ecl chemiluminescence detection kit, by Thermo Fisher Scientific (2 mentions)
Anti adipoq, by ZenBio (2 mentions)
Versadoc 4000mp system, by Bio-Rad (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Df6998, by Affinity Biosciences (1 mentions)
Df6526, by Affinity Biosciences (1 mentions)
Ab39670, by Abcam (1 mentions)
Second antibody, by Abcam (1 mentions)
Ab17026, by Abcam (1 mentions)
Ab111031, by Abcam (1 mentions)
Ab37168, by Abcam (1 mentions)
25 protocols using ripa lysis buffer
1
Western Blot Analysis of IL8 and CD274 Proteins
2
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed in cold PBS twice, then solubilized in RIPA lysis buffer (Vazyme, Nanjing, China). Samples with the same amount of protein were analyzed by western blotting as described (24 (link)).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the treated A549 and H1299 cells using RIPA Lysis Buffer (Vazyme, cat. no. FD008). And the concentration was then quantitated using Pierce BCA protein assay kit (Rockford). The equivalent proteins in each group were isolated using 10% SDS-PAGE according to their molecular weights, and the separate proteins were transferred to PVDF membrane (Millipore). After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight, followed by second antibody (Abcam) for 1 h. Finally, the membranes were treated with Enhanced ECL luminescence detection kit (Vazyme; E411-04), and the results were examined on FluorChem™ M System. The primary antibody included anti-FOXO3 (Abcam, ab17026), anti-FOXO1 (Abcam, ab39670), anti-GRB2 (Abcam, ab111031), and anti-GAPDH (Abcam, ab37168).
4
Ginsenoside Rb1, Ruscogenin, and Schisandrin Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GRS was a mixture of ginsenoside Rb1, ruscogenin and schisandrin (6:0.75:6). Schisandrin and ginsenoside Rb1 were obtained from Zelang Bio-Technology Co., Ltd (Nanjing, China). Ruscogenin was seperated in the laboratory and the purity was higher than 99%. Fetal bovine serum (FBS) was received from ScienCell (Carlsbad, CA, USA). Dulbecco’s modified Eagle medium (DMEM) was purchased from GIBCO/BRL (Life Technologies, Carlsbad, CA, USA). N-acetyl cysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from AMRESCO (Cleveland, OH, USA). The kits for determination of lactate dehydrogenase (LDH), the malondialdehyde (MDA) and the fluorescent kit for 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Protease inhibitor, RIPA lysis buffer, and enhanced chemiluminescene (ECL) reagent were obtained from Vazyme Biotech (Nanjing, China). Antibody against GAPDH was purchased from Bioworld Technology (St. Louis Park, MN, USA), antibody against JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 and Jak2, p-Jak2, Stat3, p-Stat3 were obtained from Cell Signaling Technology (Boston, MA, USA).
5
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Vazyme Biotech, Nanjing, China) with phenylmethylsulfonyl fluoride (PMSF, Roche Applied Science, Basel, Switzerland). Western blotting was performed with primary antibodies against RASSF8 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), vimentin (1:1,000, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1,000, Cell Signaling Technology), and β-actin (1:1,000, Abmart, Shanghai, China) as a loading control. The secondary antibody was goat anti-rabbit IgG conjugated with horseradish peroxidase (1:3,000, WanleiBio, Shenyang, China). Protein bands were separated and imaged using an enhanced chemiluminescence system (ProteinSimple, San Jose, CA, USA). Quantitative evaluation was conducted on Quantity One software version 4.0.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were conducted in triplicate.
6
Cellular Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Minimum essential medium (MEM), fetal bovine serum (FBS), sodium pyruvate, penicillin-streptomycin, sodium bicarbonate solution and trypsin-EDTA were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RNA Isolater Total RNA Extraction Reagent, HiScript® II Q RT Supermix for qPCR (+gDNA wiper), AceQ® qPCR SYBR®−Green Master Mix, RIPA lysis buffer and a BCA protein quantification kit were obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). The following antibodies were purchased from ProteinTech Group, Inc. (Chicago, IL, USA): JAK2 polyclonal antibody, STAT3 monoclonal antibody, and β-actin monoclonal antibody. Secondary antibodies, including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) and HRP-conjugated goat anti-mouse IgG (H+L), were obtained from Vazyme Biotech Co., Ltd.
7
Western Blot Analysis of Hrd1 and Nrf2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice in ice-cold PBS, and then solubilized in RIPA lysis buffer (Vazyme). Proteins extracted were quantified using a Bicinchoninic Acid Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Equal amounts of each protein sample (30 µg/lane) were subjected to 10% SDS-PAGE and were transferred to polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk at room temperature for 1 h. Members were incubated overnight at 4°C with antibodies targeting Hrd1 (cat. no. ab170901; 1:2,000; Abcam), Nrf2 (cat. no. sc-13032; 1:2,000; Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc.), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721; 1:3,000; Abcam) for 1.5 h at room temperature. β-actin was used as a loading control. Protein bands were visualized by enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore). ImageJ 1.45 software (National Institutes of Health) was used to perform densitometric analysis of each band.
8
Western Blot Analysis of Transfected HCC-1937 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected or untransfected HCC-1937 cells were lysed using RIPA lysis buffer (Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol. The total protein concentration was determined using a BCA Protein Quantification kit (Beyotime Institute of Biotechnology). Total protein (50 µg protein/lane) from each sample was separated via SDS-PAGE on a 12% gel and transferred onto PVDF membranes. Following blocking with 5% non-fat milk at room temperature for 2 h, all membranes were subsequently incubated with primary antibodies at 4°C overnight. Following primary antibody incubation, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (1:10,000; ab6721, Abcam) for a further 2 h at room temperature. Protein bands were visualized using ChemiGlow detection reagents (ProteinSimple). The blots were then visualized using a FluorChem 8900 Imager and semi-quantified using ImageJ software (V1.8.0; National Institutes of Health). The following primary antibodies were purchased which were obtained from Abcam: KIF18B (1:5,000; ab168812), TRIP13 (1:5,000; ab128153), MMP12 (1:10,000; ab52897), MMP9 (1:10,000; ab76003), β-catenin (1:5,000; ab32572), c-Myc (1:1,000; ab32072), cyclin D1 (1:200; ab16663) and GAPDH (1:10,000; ab181602). GADPH was used as the loading control.
9
Immunoprecipitation of NMIIA Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The treated THP-1 cells were lysed in a RIPA lysis buffer containing protease inhibitor (Vazyme Biotech, Nanjing, China). Anti-NMIIA antibody (Abcam, USA) together with protein A/G agarose, was used to immunoprecipitate NMIIA and associated proteins. Proteins were resolved by SDS-PAGE and the resulting bands were detected by Western blot analyses or stained with silver staining and then comparatively analyzed to identify specific binding proteins by MALDI-TOF-MS.
10
Protein Expression and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were lysed on ice with RIPA lysis buffer (Vazyme, FD008) for 20 min, and protein concentrations were detected with Pierce BCA Protein Assay Kit (Rockford). The protein was isolated using 10% SDS-PAGE, transferred to a PVDF membrane (Millipore), sealed with 5% skim milk for 2 h, and treated with GIGYF1 (Abcam; ab121784) and GAPDH (Abcam; ab37168), Ki-67 (Abcam; ab270650), E-cadherin (Abcam; ab233611), N-cadherin (Abcam; ab254512) overnight at 4 °C, as well as secondary antibody (Abcam; ab205719) at 37 ℃ for 1 h. Results were developed with enhanced chemiluminescence detection kit (Vazyme; E411-04) and analyzed in the FluorChem™ system.
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!