We carried out western blot analyses to detect target protein levels in kidney tissues and cells. The primary antibodies included polyclonal antibodies against phospho-YAP (Ser127) (p-YAP, 1:1000, bsm-52214R, Bioss, China), YAP (1:1000, #14074, Cell Signaling Technology, United States), α-SMA (1:10000, ab124964, Abcam, United States), collagen Ⅰ (1:750, AF7001, Affinity, United States), and GAPDH (1:1000, GB12002, Servicebio Technology, China). The primary proteins were detected by using horseradish peroxidase-conjugated secondary antibodies (Abcam, United States), and the immune complexes were finally developed by using the enhanced chemiluminescence Plus kit (Amersham, Freiburg, Germany).
Gb12002
Manufactured by Wuhan Servicebio Technology
Sourced in China
GB12002 is a freezer designed for storing biological samples at ultra-low temperatures. It has a temperature range of -86°C to -40°C and a storage capacity of 1200 liters. The product features a microprocessor-based temperature control system and an alarm function to monitor temperature changes.
Automatically generated - may contain errors
Lab products found in correlation
Gb12001, by Wuhan Servicebio Technology (4 mentions)
Gb23303, by Wuhan Servicebio Technology (2 mentions)
Ab124964, by Abcam (1 mentions)
Enhanced chemiluminescence plus kit, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Bsm 52214r, by Bioss Antibodies (1 mentions)
Af7001, by Affinity Biosciences (1 mentions)
Ecl detection system, by Beyotime (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab197202, by Abcam (1 mentions)
Ab217673, by Abcam (1 mentions)
Ab52944, by Abcam (1 mentions)
Ab52942, by Abcam (1 mentions)
Ab10433, by Abcam (1 mentions)
Sc 13156, by Santa Cruz Biotechnology (1 mentions)
Gb11190, by Wuhan Servicebio Technology (1 mentions)
Gb11132, by Wuhan Servicebio Technology (1 mentions)
Df7504, by Affinity Biosciences (1 mentions)
Gb11188, by Wuhan Servicebio Technology (1 mentions)
11 protocols using gb12002
1
Kidney Tissue Protein Expression Analysis
2
Temporal Expression of Rac1 Signaling
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To determine the temporal expression of Rac1 signaling, fresh spinal cord tissue
of the lumbar enlargement (L4-L6) was collected from deeply anesthetized rats
and then homogenized in pre-cooled lysis buffer containing a cocktail of
protease inhibitors. Total proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to a 0.2 mm
polyvinylidene difluoride membrane (Millipore, IPV H00010). The following
primary antibodies were used: anti-p-PAK1 (1:1,000, Affinity, AF3424), anti-Rac1
(1:200, Affinity, AF4200), and anti-GAPDH (1:1,000, Servicebio, GB12002).
Horseradish peroxidase-conjugated antibodies were used as secondary antibodies.
An ECL detection system (Beyotime) and ImageJ software were used to quantify the
protein bands.
of the lumbar enlargement (L4-L6) was collected from deeply anesthetized rats
and then homogenized in pre-cooled lysis buffer containing a cocktail of
protease inhibitors. Total proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to a 0.2 mm
polyvinylidene difluoride membrane (Millipore, IPV H00010). The following
primary antibodies were used: anti-p-PAK1 (1:1,000, Affinity, AF3424), anti-Rac1
(1:200, Affinity, AF4200), and anti-GAPDH (1:1,000, Servicebio, GB12002).
Horseradish peroxidase-conjugated antibodies were used as secondary antibodies.
An ECL detection system (Beyotime) and ImageJ software were used to quantify the
protein bands.
3
Protein Expression Analysis Methods
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Primary antibodies used for Western blot, immunohistochemistry and immunocytochemistry are listed as followed: Hif-1α (79233, Cell signaling, USA), α-SMA (ab32575, Abcam, USA), Bnip3 (ab10433, Abcam, USA; ab38621, Abcam, USA), LC3B (83506, cell signaling, USA; 12741, cell signaling, USA), vimentin (ServiceBio, GB12192, China), phosphor-vimentin Ser56 (ab217673, Abcam, USA), phosphor-vimentin Ser38 (ab52942, Abcam, USA), phosphor-vimentin Ser72 (ab52944, Abcam, USA), phosphor-vimentin Ser82 (ab52943, Abcam, USA), caspase 3 (ab197202, Abcam, USA), cytochrome C (sc-13156, Santa Cruz, USA), GAPDH (GB12002, Servicebio, China).
4
Protein Expression Analysis by Western Blot
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The cell samples were ultrasonically treated in lysis buffer on ice, and the supernatant was collected after 12,000 rpm centrifugation for 10 min at 4 °C. The protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit. Proteins were loaded onto an SDS-polyacrylamide gel electrophoresis (PAGE) gel, and then transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with blocking buffer for 2 h at room temperature, and incubated with specific primary antibodies against VEGFR2 (Servicebio, Shanghai, China; GB11190) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Servicebio, Shanghai, China; GB12002) overnight at 4 °C. Next, membranes were incubated with secondary horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) antibody (Servicebio, Shanghai, China; GB23303) for another 1 h at room temperature after washing. PVDF membranes were exposed using a Tanon 4200SF Chemiluminescent Imaging System (Shanghai Tanon Science & Technology Co., Ltd., Shanghai, China).
5
Rat model of blood-brain barrier dysfunction
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JFG (0012007009) was bought from Shandong New Times Pharmaceutical Co., Ltd. Primary antibodies of anti-β-actin (GB12001, Servicebio, Wuhan, China), anti-GAPDH (GB12002, Servicebio, Wuhan, China), anti-MMP9 (GB11132, Servicebio, Wuhan, China), anti-PI3K (GB11525, Servicebio, Wuhan, China), anti-AKT (GB13427, Servicebio, Wuhan, China), anti-p-AKT (AF0016, Affinity Biosciences, USA), anti-IL-1β (AF5103, Affinity Biosciences, USA), anti-Occludin (DF7504, Affinity Biosciences, USA), anti-Claudin-5 (AF5216, Affinity Biosciences, USA), and anti-TNF-α (GB11188, Servicebio, Wuhan, China). Evans Blue (CAS: 314-13-6) was purchased from Bioengineering (Shanghai) Co., Ltd. (Shanghai, China). The Collagenase IV (C8160) and rabbit fluorescein conjugated antibody were purchased from Servicebio (Wuhan, China). Adult male rats (Sprague-Dawley rat, 300 ± 20 g) were purchased from Pengyue Laboratory Animal Breeding Co., Ltd. (Jinan, Shandong, China).
6
Protein Extraction and Western Blot Analysis
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The tissues were collected and homogenized immediately, and the protein was extracted using the Total Protein Extraction Kit. The BCA protein assay kit was used to determine the protein concentration. Twenty micrograms total protein was loaded and separated on a 10% SDS poly-acrylamide gel, and then transferred to the PVDF membrane (Roche). The membrane was blocked with 5% non-fat dry milk for 2 h, followed by incubation with primary antibodies against α-SMA (1:500, 1A4, DAKO) or GAPDH (1:500, GB12002, Servicebio) overnight. Then the membrane was incubated with the secondary antibody. The specific signals were detected by the Immobilon Western Chemiluminescent HRP Substrate (Millipore) and Tanon image system.
7
Western Blot Analysis of ECM Proteins
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NIH 3T3 cells were treated as described previously. At the end of incubation, cells were collected and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail. The concentration of protein was assessed by NanoDrop spectrophotometer. Then loaded the protein (200 μg) on SDS-PAGE (10% or 12%) and blotted onto NC membranes. After blocking, the primary antibodies were incubated with membranes overnight at 4 °C to detect the specific protein. Anti-α-SMA (14395-1-AP, Proteintech), anti-Collagen 1 (14695-1-AP, Proteintech), and anti-GADPH (GB12002, Servicebio) antibodies used in this part were configured at a concentration of 1:1000. The HRP-conjugated secondary antibodies against mouse (1:3000, GB23301, Servicebio) or HRP-conjugated secondary antibodies against rabbit (1:3000, GB23303, Servicebio) were used to detected appropriate primary antibodies. Bands were visualized with ECL-system, and images were captured using the chemiluminescence instrument.
8
Murine Tissue Protein Analysis
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Protein was extracted from murine tissues and THP-1-derived macrophages of different phenotypes for western blot. The blots were probed with antibodies against IL-6 (DF6087, Affinity Bioscience), FCN1 (10930-R017, Sino Biological), IL-1β (12242S, CST), GAPDH (GB12002, Servicebio), β-actin (GB12001, Servicebio), caspase-1 (3866S, CST), cleaved caspase-1 (4199S, CST), and NLRP3 (15101S, CST).
9
Immunoblot Analysis of Protein Expression
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Animal tissues were homogenised and incubated for 60 min at 4℃ in lysis buffer and separated through SDS/PAGE for Western blot analyses. Primary antibodies included: Ghrelin (Abcam #ab129383), growth hormone secretagogue receptor-1α (GHS-R1α; Abcam #ab95250), protein kinase B (Akt; CST #4691), p-Akt (CST #4060), mammalian target of rapamycin (mTOR; CST #2983), p-mTOR (CST #5536), muscle ring-finger1 (MURF-1; Abcam #ab172479), muscle atrophy F-box (MAFBx; Abcam #ab168372), MUC2(Abcam #ab272692), ZO-1(WUHAN SANYING #21773-1-AP),GAPDH(Servicebio #GB12002) and β-actin (Servicebio #GB12001).
10
Western Blot Analysis of Uterine and Cell Markers
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The uterine horns and Ishikawa and HESCs were separately prepared using RIPA (G2002, Servicebio, China) with PMSF (G2008, Servicebio, China). Protein concentration was quanti ed using BCA kit (Cat#P0010S, Servicebio, China). Aliquots of protein samples were separated by SDS-PAGE (G2003, Servicebio, China). The separated proteins were subsequently blotted on to PVDF membrane (G2008G6015-0.45, Servicebio, China) and incubated with primary antibodies to MELTF (1:1000, Cat No.10428-1-AP, Proteintech, China), primary antibodies to LC3B (1:1000, Cat No.18725-1-ap, Proteintech, China), primary antibodies to NLRP3 (1:1000, WL02635, Wanlei, China), primary antibodies to ACSL4 (1:1000, GB113871, Servicebio, China), primary antibodies to FPN1 (1:1000, GB11147, Servicebio, China), primary antibodies to GPX4 (1:1000, Cat No.67763-1-lg, Proteintech, China), primary antibodies to β-actin (1:2000, GB12001, Servicebio, China) or GAPDH (1:2000, GB12002, Servicebio, China). The membrane was then washed and treated with peroxidase-conjugated secondary anti-rabbit antibody (1:5000, GB25301, Servicebio, China) or peroxidase-conjugated secondary anti-mouse antibody (1:5000, GB25301, Servicebio, China).
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