Chicken type II collagen and complete Freund adjuvant were purchased from Chondrex. H&E staining kit was from Jiancheng Bio. Safranine red and Fast Green staining kit was from Solarbio. Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco. Fetal bovine serum (FBS) was from Wisent. Diaminobenzidine peroxidase substrate kit was from Vector Laboratories. Transfection reagent GenJet was from SignaGen. Bicinchoninic acid protein assay kit was from Thermo Fisher. Enhanced chemiluminescence detection kit was from Vazyme. Cytokines such as TNF-α, IL-1β, IL-6, and LPS were from Peprotech. Nuclear and cytoplasmic extraction reagents were from Thermo Fisher. The antibody against DEC1 was described elsewhere (61 (link)). Anti-CTSK was from Santa Cruz Biotechnology. Anti-NFATc1 was from Cell Signaling Technology. All other antibodies were from BioGot. Human IL-6 ELISA Kit was from SHRBIO.
Enhanced chemiluminescence detection kit
Manufactured by Vazyme
Sourced in China
The Enhanced Chemiluminescence Detection Kit is a laboratory tool designed to facilitate the detection and analysis of proteins in western blot experiments. The kit provides reagents for the generation and amplification of chemiluminescent signals, which can be used to visualize and quantify target proteins.
Automatically generated - may contain errors
Lab products found in correlation
N cadherin, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti ctsk, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Diaminobenzidine peroxidase substrate kit, by Vector Laboratories (1 mentions)
Complete freund adjuvant (cfa), by Chondrex (1 mentions)
Anti nfatc1, by Cell Signaling Technology (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Chicken type 2 collagen, by Chondrex (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
Genjet, by SignaGen (1 mentions)
Fibronectin, by Abcam (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
10 protocols using enhanced chemiluminescence detection kit
1
Murine Osteoarthritis Induction and Analysis
2
Protein Expression and Glycosylation Analysis
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Total proteins (30 μg) from samples were separated by 7.5% SDS‐PAGE. Gels were transferred onto PVDF membranes using Trans‐Blot Turbo Transfer System (Bio‐Rad Laboratories). Membranes were soaked in 5% skim milk in TBST (20 mM Tris‐HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) for 2 hours at 37°C, probed with primary antibodies directed to FUT8 (1:500; cat # sc271244), AP‐2γ (1:500; cat # sc12762; Santa Cruz Biotechnology), N‐cadherin (1:5000; cat # ab18203), GAPDH (1:5000; cat # ab8245), fibronectin (1:1000; cat # ab2413; Abcam), E‐cadherin (1:10 000; cat # 610181; BD Biosciences), AKT (pan) (1:1000; cat # 4685), p‐AKT (Ser473) (1:2000; cat # 4060), STAT3 (1:1000; cat # 9139), and p‐STAT3 (Tyr705) (1:1000; cat # 9145; Cell Signaling Technology) overnight at 4°C, and incubated with appropriate HRP‐conjugated secondary antibody. Specific bands were visualized using Pro‐light HRP Kit (Tiangen). For lectin blotting, biotinylated lectins Lens culinaris agglutinin (LcH) and Aleuria aurantia lectin (AAL) were incubated after blocking by 3% (w/v) bovine serum albumin (BSA) in PBST (PBS with 0.5% [v/v] Tween 20), and bands were detected using VECTASTAIN ABC kits. Bands were visualized using enhanced chemiluminescence detection kit (Vazyme) and imaged with ChemiDoc XRS+ (Bio‐Rad).
3
Protein Expression Analysis by Western Blotting
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Equal amounts of protein lysates (40 μg) were analyzed by SDS-PAGE and were electrotransferred to polyvinylidene difluoride membranes. The membranes were washed with 1X Tris-buffered saline 1% Tween-20 (TBST), blocked for 1 h at room temperature (RT) in 5% milk/TBST, and immunoblotted with specific antibodies, which included the rabbit anti-SLC25A38, anti-Notch1 (Abcam) and anti-GAPDH (Cell Signaling Technology, Inc.) monoclonal antibodies. GAPDH served as a loading control. On the subsequent day, the membranes were washed with 1X TBST and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies diluted to 1:4,000 in 5% milk/TBST for 1 h at RT. The antibodies were detected using an enhanced chemiluminescence detection kit (Vazyme Biotech Co., Ltd., Nanjing, China). The relative densities of the presenting quantity of target proteins were assessed using Quantity One software (Bio-Rad) followed by normalization against the housekeeping protein, GAPDH.
4
Western Blot Analysis of Protein Markers
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As the previously described [23 (link)], RIPA Lysis buffer (Beyotime, Shanghai, China) was used to extract total protein. Equal amount of protein was separated using 10% SDS-PAGE gel and then electro-transferred onto PVDF membranes (Invitrogen). The membranes were blocked with 5% nonfat milk followed by incubated overnight with primary antibodies at 4°C, including CD9 (1:2,000, ab92726, Abcam, Cambridge, MA, USA), CD63 (1:1,000, ab134045, Abcam), CD81 (1:2,000, ab109201, Abcam), ZO-1 (1:1,000, ab216880, Abcam), occludin (1:250, ab31721, Abcam), E-cadherin (1:10,000, ab40772, Abcam), α-SMA (1:1,000, ab5694, Abcam), Bcl-2 (1:1,000, ab194583, Abcam), Bax (1:1,000, ab32503, Abcam), cleaved caspase 3 (1:1,000, ab2302, Abcam), and β-actin (1:1,000, ab5694, Abcam). After further incubated with secondary antibody (1:50,000, ab205718, Abcam), the protein signals were visualized using Enhanced Chemiluminescence Detection Kit (Vazyme, Nanjing, China).
5
Protein Isolation and Western Blot Analysis
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Total protein was isolated from serums, cells and exosomes using RIPA buffer (Beyotime) and quantified using BCA protein assay kit (Tiangen, Beijing, China). Then, equal amount of proteins was separated by sodium dodecyl sulfonate–polyacrylamide gel (Solarbio) and transferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, New York, NYC, USA). After being blocked with skim milk for 2 h, membranes were incubated with primary antibodies against multidrug resistance-associated protein 1 (MRP1; ab32574; Abcam, Cambridge, MA, USA), multidrug resistance protein 1 (MDR1; ab170904; Abcam), REV3L (ab111729; Abcam) or GAPDH (ab181602; Abcam) overnight followed by incubation with corresponding secondary antibody (ab150077; Abcam) for 2 h. The proteins were visualized using the enhanced chemiluminescence detection kit (Vazyme, Nanjing, China).
6
Western Blot Analysis of Oxidative Stress Markers
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Proteins from each group of cells were extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime). The protein concentrations were determined using a BCA protein assay kit (Vazyme). Equal amounts (10 μg) of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk buffer for 1 h at room temperature, and then incubated with primary antibodies against HO-1, Nrf2, or GAPDH (1:1000; Proteintech) overnight at 4 °C. The membranes were washed three times, and then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10000; Proteintech) for 1 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence detection kit (Vazyme), and protein quantification was performed using ImageJ 1.8.0 software.
7
Protein Extraction and Western Blot Analysis
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Cells and liver tissues were treated with ice-cold RIPA buffer containing newly added PMSF solution (Beyotime, #ST507) and phosphatase inhibitors (KeyGEN, #KGP602) to extract proteins. BCA assay was then conducted to determine the concentrations. Subsequently, the protein samples were subjected to SDS‒PAGE for separation, followed by transfer onto a PVDF membrane (Bio-Rad). Immunoblotting was performed by blocking the membrane with 5% BSA in TBST buffer, incubating with specific primary antibodies, and then with appropriate secondary antibodies. Protein visualization was achieved using an enhanced chemiluminescence detection kit (Vazyme, #E412-01). The pertinent details regarding the antibodies used in Western blot can be found in Supplementary Table 4 .
8
Western Blot Analysis of Lung Tissue
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Western blot analysis was performed as previously described (Shi et al., 2022 (link)). Briefly, lung tissue was ground at low temperature and the protein concentration was measured with bicinchoninic acid (BCA) kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). Proteins (15 μg) were separated by sodium dodecyl sulfate-polyacrylamide gen electrophoresis and transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat dried milk in Tris-Buffered Saline with 0.1% Tween 20 (TBST) for 1–2 h at room temperature. After washing three times with TBST, the membranes were incubated with primary antibodies (1:1,000) overnight at 4°C. The membrane was washed three times with TBST and then incubated with secondary antibodies (1:5,000) for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection kit (Vazyme Biotech Co., Ltd., Nanjing, China).
Antibodies for the detection of P38 (8690S), p-P38 (4511S), Erk1/2 (4695S), p-Erk1/2 (4370S), AKT (4691S), p-AKT (4060S), PI3K (4249S), β-actin (3700S) and p-PI3K (17366S) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, United States).
Antibodies for the detection of P38 (8690S), p-P38 (4511S), Erk1/2 (4695S), p-Erk1/2 (4370S), AKT (4691S), p-AKT (4060S), PI3K (4249S), β-actin (3700S) and p-PI3K (17366S) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, United States).
9
Protein Expression and Western Blot Analysis
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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.
10
Modulation of MDSC Phenotype by Doxorubicin
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The MDSCs were sorted from the bone marrows of the B16-F10 tumor-bearing mice and cultured in MDSC culture medium. The cells were divided into three groups, namely, control, DOX-low (2.5 μM), and DOX-high (5 μM) groups. After the cells were treated with DOX for 48 h, the expression of iNOS and Arg-1 in MDSCs was determined by Western blot analysis. After 24 h, the cells were treated with drugs for 24 h or 48 h, washed with PBS, and lysed in ice-cold lysis buffer with protease inhibitor cocktail (Sigma Aldrich, Saint Louis, U.S.A) for 30 min. The lysates were separated through SDS-PAGE and then transferred to PVDF membranes (Millipore, Massachusetts, USA). These membranes were then blocked and incubated first with primary antibodies Arg-1 (1:1000, Affinity Bioreagents, Colorado, U.S.A) and iNOS (1:1000, Affinity Bioreagents, Colorado, U.S.A) and subsequently with secondary antibodies (1:5000, Affinity Bioreagents, Colorado, U.S.A). The blots were visualized with an enhanced chemiluminescence detection kit (Vazyme, Nanjing, China).
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