Protein was isolated from VSMCs or aortic tissues using a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., USA). The protein content was determined in the supernatants using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., USA). Protein lysates (30 μg/sample) were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore Corp., USA) membranes. The membranes were blocked in 5% fat-free milk overnight at 4°C. Membranes were then probed with the following primary antibodies purchased from Abcam (Cambridge, USA): anti-α-SMA (cat. no. ab5694, 1:1,000), anti-OPN (cat. no. ab8448, 1:1,000), anti-MMP-2 (cat. no. ab92536, 1:1,000), anti-MMP-9 (cat.no. ab38898, 1:1,000), and anti-TIMP-1 (cat. no. ab38978, 1:1,000) and incubated overnight at 4°C. Subsequently, protein bands were detected by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG, cat. no. ab6789; 1:2,000; Abcam) at room temperature for 1 h. Signals were detected using an enhanced chemiluminescence kit (ECL kit, Wuhan Boster Biotechnology Co., Ltd, China) and exposed to Kodak X-OMAT film (Kodak, Rochester, USA). The band intensity was quantified with the Quantity One software. Each experiment was performed at least three times. GAPDH served as the loading control.
Ripa lysis buffer kit
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The RIPA lysis buffer kit is a cell lysis solution designed for the extraction and solubilization of proteins from mammalian cells and tissues. It contains a balanced mixture of ionic and non-ionic detergents, as well as other components, to facilitate the efficient disruption of cell membranes and the release of soluble cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay, by Bio-Rad (7 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ezq protein quantitation kit, by Thermo Fisher Scientific (3 mentions)
X omat film, by Kodak (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tgf β1, by Abcam (2 mentions)
Ecl detection system, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Anti α sma, by Abcam (1 mentions)
Mmp13, by Abcam (1 mentions)
Col2a1, by Abcam (1 mentions)
Smad3, by Abcam (1 mentions)
Instantone elisa, by Thermo Fisher Scientific (1 mentions)
El800 elisa reader, by Agilent Technologies (1 mentions)
Collagen 3, by Abcam (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Collagen 1, by Abcam (1 mentions)
Tmt 6plex isobaric labeling reagents, by Thermo Fisher Scientific (1 mentions)
21 protocols using ripa lysis buffer kit
1
Western Blot Analysis of Vascular Smooth Muscle Cell Proteins
2
Brain Protein Extraction using RIPA Lysis Buffer
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Whole cell protein was extracted from brain tissue using the RIPA Lysis Buffer Kit (Santa Cruz Biotechnology, Inc. Santa Cruz, CA.) according to the manufacturer's protocol. Briefly, an appropriate amount of RIPA complete lysis buffer was added to cell pellet. The mixture was incubated on ice for 5 minutes, followed by centrifugation at 14000× g for 15 min at 4°C. The supernatant was collected as brain tissue lysate and stored at −80°C until further use. Protein concentration was determined using Invitrogen EZQ Protein Quantitation Kit (Invitrogen, Grand Island, NY). Protein from all mice (n = 157), spanning all time points, was pooled as reference material.
3
Western Blot Analysis of Chondrocyte Proteins
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Total protein was extracted from chondrocytes using a RIPA lysis buffer kit
(Santa Cruz, TX, USA). Protein lysates were separated on 10% SDS-PAGE and
transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp.
Billerica, MA, USA). After being blocked in 5% fat-free milk overnight at 4°C,
the membranes were then incubated with the following primary Abs: MMP-13
(1:1000, Abcam, UK), TGF-β1 (1:1000, Abcam), SMAD3 (1:1000, Abcam), CTGF
(1:1000, Abcam) and COL2A1 (1:1000, Abcam) at 4°C overnight. Then the membranes
were incubated with the secondary Ab IgG at room temperature for 1 h. The band
intensity was quantified with software Image J 6.0. GAPDH was used as the
loading control.
(Santa Cruz, TX, USA). Protein lysates were separated on 10% SDS-PAGE and
transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp.
Billerica, MA, USA). After being blocked in 5% fat-free milk overnight at 4°C,
the membranes were then incubated with the following primary Abs: MMP-13
(1:1000, Abcam, UK), TGF-β1 (1:1000, Abcam), SMAD3 (1:1000, Abcam), CTGF
(1:1000, Abcam) and COL2A1 (1:1000, Abcam) at 4°C overnight. Then the membranes
were incubated with the secondary Ab IgG at room temperature for 1 h. The band
intensity was quantified with software Image J 6.0. GAPDH was used as the
loading control.
4
NF-κB Pathway Activation in HCMV-Infected Macrophages
5
Protein Expression Analysis of Cardiac Fibrosis
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Briefly, total protein was extracted from atrial muscle tissues of the patients, human atrial fibroblasts, or mouse atrial muscle tissues using a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Protein lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp. Billerica, MA, USA). After being blocked in 5% fat-free milk overnight at 4 °C, the membranes were then incubated with the following primary antibodies: Collagen I (1:1000; Abcam, Cambridge, MA, USA), Collagen III (1:1000; Abcam), TGF-β1 (1:1000; Abcam), Smad2 (1:1000; Cell Signaling Technology Inc.), Smad3 (1:1000; Cell Signaling Technology Inc.), and Sp1 (1:1000; Cell Signaling Technology Inc.) at 4 °C overnight. Then the membranes were incubated with the secondary antibody IgG at room temperature for 1 h. The band intensity was quantified with software Quantity One. β-actin served as the loading control.
6
Quantitative Proteomics of Genital Tract
7
Western Blot Protein Expression Analysis
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Cells were lysed on ice using RIPA Lysis buffer kit with protease inhibitor cocktail (Santa Cruz Biotechnology Inc., Heidelberg, Germany). Protein concentration was measured using a BCA protein assay kit (Pierce, Thermo Scientific, Rockford, U.S.A). Thirty μg of reduced proteins in Laemmli sample buffer (Biorad, Segrate, Milan, Italy) were resolved using Gel precast Miniprotean 4-20% (Biorad) and transferred to nitrocellulose by Semi-Dry Trans-Blot Turbo System (Biorad). Membranes were blocked with 5% non-fat dry milk (Biorad) in TBS-T, and incubated with primary antibody overnight at 4°C and thereafter with the appropriate HRP-conjugated secondary antibody (Biorad) for 1h at room temperature. Membranes were developed using ECL detection reagent (Amersham, Glattbrugg, Switzerland). The primary antibodies were: anti-EP-CAM, anti-E-cadherin, anti-Vimentin (1:1000) and anti-α-Tubulin (1:2000) (Cell Signalling Technology, Massachusetts, USA). Blots were stripped for 20 minutes using the stripping buffer (Thermo Scientific) before re-probing.
8
Protein Expression Analysis in Tissue and Cells
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Total proteins were extracted from tissue samples and cultured cells using a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., USA). Approximately equal amounts of protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with specific primary antibodies including anti-AXL (1:500, Abcam, USA), anti-N-cadherin (1:500, Abcam, USA), anti-E-cadherin (1:500, Abcam, USA), anti-Vimentin (1:500, Abcam, USA) and anti-GAPDH (1:500, Abcam, Cambridge, Britain) at 4°C overnight. On the following day, the membranes were incubated with appropriate HRP-linked secondary antibodies (1:10,000, Sigma) at room temperature for 2 h. The protein bands were visualized by enhanced chemiluminescence (ECL) kit (Takara), and the intensity of targets were analyzed using the Quantity One software.
9
Western Blot and Autophagic Flux Analysis
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Western blot analysis was performed as described earlier [26 (link), 41 (link)]. Briefly, protein lysates were prepared using the RIPA Lysis Buffer Kit (Santa Cruz Biotechnology), according to the manufacturer's protocol. Electrophoresis and transfer of proteins were performed using standard methods and protein–antibody complexes were detected by chemiluminescence assays. Quantitation of the signal was performed using Bio-Rad Image Lab analysis software. The autophagic flux assay was performed using saturating concentrations (20-40 nM) of lysosomal inhibitor Bafilomycin A1.
10
Western Blot Analysis of Protein Expression
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Protein was collected and lysed in radioimmunoprecipitation buffer (RIPA) containing protease inhibitors at 4°C for 30 min. Cell lysates were prepared with a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., Calif., USA), and the protein concentrations were quantified using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc.). Proteins (30 μg) were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Amersham; GE Healthcare, Chicago, IL, USA). The membranes were blocked in 5% nonfat milk (Merck KGaA, Darmstadt, Germany) overnight at 4°C. Transferred membranes were then stained with the primary antibodies overnight at 4°C. Subsequently, protein bands were detected by incubation with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The immune reactive proteins were visualized using SuperSignal ECL (Pierce, Rockford, IL, USA) and the densities of the bands were analyzed with Image J (NIH, Bethesda, MD, USA).
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