Cells and tissue lysates were obtained utilizing RIPA buffer (Beyotime, Shanghai, China), and quantified by the BCA method. The obtained protein samples (40 μg) were size-fractionated on SDS-PAGE gels and then transferred to PVDF membranes. After hindered non-specific binding, the membranes were probed with anti-NECAB3 (PA5-101422, 1:1,000, Thermo Fisher Scientific), HIF-1α (ab216842, 1:1,000, Abcam), RIT1 (ab53720, 1:1,000, Abcam), and actin (ab200658, 1:5,000, Abcam) antibodies, and then incubated with secondary antibody IgG H&L (HRP) (ab6721, Abcam, Cambridge, MA, UK). Eventually, the bands were observed through using the ECL detection system (Bio-Rad, Hercules, CA, USA).
Ab216842
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab216842 is a laboratory equipment product. It functions as a core instrument for research and analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Ab137867, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab11672, by Abcam (2 mentions)
Exo fect exosome transfection reagent, by System Biosciences (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab200658, by Abcam (1 mentions)
Igg h l hrp, by Abcam (1 mentions)
Ecl detection system, by Bio-Rad (1 mentions)
Ab92337, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
Ab1316, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Ab76319, by Abcam (1 mentions)
Fg 4592, by Selleck Chemicals (1 mentions)
17 protocols using ab216842
1
Western Blot Analysis of Protein Expression
2
Quantifying Angiogenic Protein Levels
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VEGFA, HIF1α, FGF2, and CXCL12 protein levels in ADSCs and tissues were determined by western blotting as previously described [11 (link), 12 (link)]. Briefly, after electrophoresis and transfer to polyvinylidene difluoride membranes, proteins were incubated with antibodies against HIF1α (ab216842, 1:500, Abcam, Cambridge, MA, USA), anti-VEGFA (ab1316, 1:100, Abcam), FGF2 (ab92337; 1:300; Abcam), CXCL12 (ab137867; 1:300; Abcam), and GAPDH (#5174; 1:20,000; Cell Signaling Technologies, Danvers, MA, USA) at 4 °C overnight. Proteins were visualized using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) and exposure to X-ray film and quantified by laser scanning densitometry (GE Healthcare Life Sciences, Piscataway, NJ). GAPDH was used as a loading control.
3
Exosome Isolation and Characterization Protocol
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Dulbecco's Modified Eagle Medium (DMEM, high glucose, 11965092), DMEM/F12 and Fetal bovine serum (FBS, 10100147) was obtained from Gibco (USA). DPBS, 1% penicillin/streptomycin solution and 0.25% trypsin‐EDTA were obtained from HyClone (USA). Exosome‐depleted FBS was obtained from VivaCell (Israel, C3801‐0100). Si‐RNA transfection reagent was Advanced Transfection Regeant (Zeta Life, USA). The reagent transferring Si‐RNA into exosomes was Exo‐Fect Exosome Transfection Reagent (SBI, EXFT20A‐1). DID was purchased from beyotime (C1995S). The following antibodies were used in this study: mouse anti LAMP2 antibody (1:1000 for WB; Proteintech, 66301‐1‐Ig), rabbit anti‐TSG101, (1:1000 for WB; Proteintech, 28283‐1‐AP), rabbit anti‐CD63 (1:1000 for WB; Proteintech, 25682‐1‐AP), rabbit anti‐CD81 (1:1000 for WB; Proteintech, 66866‐1‐Ig), mouse anti‐β‐actin (1:1000 for WB; Proteintech, 66009‐1‐Ig), rat anti‐F4/80 antibody (1:100 for IF; Abcam, ab6640), rabbit anti‐iNOS (1:100 for immunofluorescence; Proteintech,18985‐1‐AP), rabbit anti‐HIF‐1α (1:100 for IHC‐P; Abcam, ab216842).
4
Mitochondrial Dynamics and Inflammation Markers
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Rabbit anti-Mfn1 (14739s), rabbit anti-Opa1 (67589s), rabbit anti-Fis1 (86668s), rabbit anti-Drp1 (8570s), rabbit anti-IL-1β (12703s), rabbit anti-CD3 (26582s), rabbit anti-Myeloperoxidase (MPO+) (14569T), rabbit anti-F4/80 (30325s), and rabbit anti-collagen I (72026T) antibodies were taken from CST; mouse anti-β-actin (ab8226), rabbit anti-HIF-1α (ab216842), mouse anti-PCNA (ab29), rabbit anti-E-cadherin (ab76319), rabbit anti-NCC (ab203674), rabbit anti-α-SMA (ab5694), mouse anti-vimentin (ab92547), rabbit anti-GPX-4 (ab125066), and rabbit anti-fibronectin (Fn) (ab2413) antibodies were obtained from Abcam; rabbit anti-TNF-α (7B8A11) antibody was purchased from Proteintech; rabbit anti-ZO-1 (PA524716), rabbit anti-AQP1 (101AP), and rabbit anti-AQP2 (201AP) antibodies were obtained from Invitrogen; anti-IL-33 (AF3626) antibody was acquired from R&D Systems. FG-4592 was obtained from Selleck and FA was acquired from Dalian Meilun Biotechnology Co. of China.
5
Protein Expression Analysis in CRC
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Twenty-five micrograms of the total proteins in CRC tissues and cell lines that were harvested was loaded and separated by 10% SDS-PAGE gel. The cytosolic fraction and nuclear extracts were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) following the manufacturer's protocol. Isolated proteins were moved to PVDF membranes (88518, Thermo Scientific, USA) followed by 1-h blocking at room temperature in 5% nonfat milk (LP0033B, Thermo Scientific, USA) contained in 1 × TBST buffer (28360, Thermo Scientific, USA). Next, the membranes were incubated at 4°C with primary antibody for a night (RIG-I, ab180675; HIF-1α, ab216842; PKM2, ab85555; LDHA, ab52488; Lamin B1, ab229025; and β-actin, ab8226 all from Abcam, UK; GLUT1, Biorbyt, St Louis, MO, USA, orb157188; NF-κBp65, Cell Signaling Tech, #8242). Lastly, 1 × TBST was employed to wash the above membranes twice, and ECL (WBULS0100, Millipore, USA) was used to treat them for visualizing specific protein bands. The quantitative analysis for each protein band was performed by ImageJ software (USA).
6
Quantitative Protein Analysis in Cells
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Total protein was extracted from cells by incubation in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 1 mM phenylmethanesulfonyl fluoride and 20 mM N-ethylmaleimide, as previously reported (19 (link)). Then, 100 µg proteins were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (EMD Millipore, Inc.). Then, the membrane was blocked in 5% BSA (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h and incubated with the specific antibodies against SOX2 (ab137385; Abcam; 1:1,000), Oct4 ab18976; 1:1,000), SUMO1(ab11672; Abcam; 1:1,000), HIF-1α (ab216842; Abcam; 1:500), vimentin (5741; Cell Signaling Technology, Inc.; 1:1,000), E-cadherin (610404; BD Biosciences; 1:500) and β-actin as an internal control (ab8227; Abcam; 1:1,000). The gray value of the bands was quantified by ImageJ image analysis software, version 1.48 (National Institutes of Health).
7
Dexamethasone Regulates HCC Stem Cell Markers
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8
Western Blot Analysis of Protein Markers
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Protein level in lysis supernatant of BxPc‐3 cells was determined by bicinchoninic acid (BCA) protein assay kit (Thermo), and 25 μg of which was separated on 10% to 15% of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Electrophoretic pure of HIF‐1α, LOXL2, E‐cadherin, MMP2, and MMP9 were transferred onto nitrocellulose (NC) membranes (Millipore), and incubated with antibody against HIF‐1α (abcam, Ab216842), anti‐LOXL2 antibody (abcam, Ab96233), antibody against E‐cadherin (Cell Signaling Technology [CST], #14472), antibody against MMP2 (Abcam, Ab14311), antibody against MMP9 (Abcam, Ab137867), anti‐β‐actin antibody (Abcam, ab8226), and anti‐GAPDH antibody (CST, #5174) at 4°C overnight followed by horseradish peroxidase‐conjugated antibodies (Beyotime, A0208, Shanghai, China) for another 1 hour at 25°C. Immunoreactive bands were qualified by enhanced chemiluminescence (ECL) system (GE Healthcare/Amersham Biosciences). We chose β‐actin as a “loading” control (instead of GAPDH) in hypoxic studies, accordingly to a reported study.19
9
Cardiac Tissue Immunohistochemistry Protocol
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Cardiac tissues were fixed in 4% paraformaldehyde, cut into slices, and embedded with paraffin. After dewaxing, slides were batched for 20 min at 80°C, and then incubated with TRFC antibody (ab214039; Abcam), α‐actinin antibody (ab90421; Abcam), F4/80 antibody (aab6640; Abcam), Hif‐1α antibody (ab216842; Abcam), at 4°C overnight, and treated with fluorescence‐conjugated secondary antibody for 1 h at 37°C. The nuclei were stained with DAPI. After washing, the sections were imaged by a fluorescence microscope.
10
Lung Tissue Protein Expression Analysis
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Protein expression in total lung tissues, HPMECs, and PASMCs were determined by Western blotting. After transferation, the PVDF membranes (Millipore, United States) were blocked with 5% skim milk to prevent non-specific binding, then incubated with specific primary antibodies against HIF1-α (1:500, ab216842, Abcam, United States), NOX4 (1:1,000, 14347-1-AP, Proteintech, China), SOD2 (1:1,000, 24127-1-AP, Proteintech, China), SOD1(1:1,000, 10269-1-AP, Proteintech, China), Cleaved Caspase-3 (1:1,000, 9664, Cell Signaling Technology, United States), Bcl-xL (1:1,000, AB126, Beyotime, China), β-actin (1:2,500, 60008-1-Ig, Proteintech, China). After washing with TBST buffer, the membranes were incubated with appropriate secondary antibody conjugated with horseradish peroxidase, and signals were detected using by enhanced chemiluminescent kit (Amersham Biosciences, United Kingdom). The relative OD of Western blotting was calculated with the Image lab software (Bio-Rad Laboratories, Hercules, CA, United States).
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