At 48 h after transfection, cells in each group were collected. Total protein was extracted from cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined with an enhanced bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 40 µl of protein was loaded and subjected to 10% SDS-PAGE. The separated protein was then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk at 37°C for 1 h, the PVDF membranes were incubated with primary antibodies against CBL (1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog number, sc-170) and β-actin (1:2,000 dilution; Santa Cruz Biotechnology, Inc.; catalog number, 4970) at 4°C overnight. After washing with Tris-buffered saline containing Tween-20, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:5,000 dilution; Beyotime Institute of Biotechnology; catalog number: A0208) at room temperature for 60 min. The target bands were visualized using an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Relative protein levels of CBL were calculated using β-actin as an internal reference.
Horseradish peroxidase labeled secondary antibody
Manufactured by Beyotime
Sourced in China
Horseradish peroxidase-labeled secondary antibody is a laboratory reagent used in various immunoassays and detection techniques. It consists of a secondary antibody conjugated with the enzyme horseradish peroxidase. The enzyme catalyzes a colorimetric reaction, enabling the visualization and quantification of target analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Bcl 2, by Abcam (2 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Rabbit anti β actin, by Beyotime (2 mentions)
Polyvinylidene uoride membrane, by Merck Group (2 mentions)
Enhanced bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride blotting membrane, by GE Healthcare (1 mentions)
Enhanced chemiluminescence solution kit, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Molecular imager, by Bio-Rad (1 mentions)
Neuronal nuclei (neun), by Cell Signaling Technology (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
Anti gfap, by Abcam (1 mentions)
27 protocols using horseradish peroxidase labeled secondary antibody
1
Western Blot Analysis of CBL Expression
2
Protein Isolation and Western Blot Analysis
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Proteins were isolated from RAW264.7 cells by using radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). A BCA Protein Assay kit (Beyotime) was applied to measure the protein concentration (562 nm) compared with a protein standard. Sample proteins then were electrophoresed on 4–12% sodium dodecyl sulfate‐polyacrylamide gels and then transferred to a polyvinylidene fluoride blotting membrane (GE Healthcare, Fairfield, CT, USA). The membranes were blocked in 5% non‐fat dry milk with 1× Tris‐buffered saline with Tween 20 (TBST) for 1 h at room temperature and incubated with the primary antibodies overnight at 4°C. Subsequently, the membranes were treated with horseradish peroxidase‐labeled secondary antibodies (Beyotime) for 1 h at room temperature. The detection of protein expression was visualized by an enhanced chemiluminescence solution kit (Millipore, Billerica, MA, USA) in a molecular imager (Bio‐Rad, Herculas, CA, USA). Quantification of relative changes in protein levels was carried out by Image Lab software (Bio‐Rad).
3
Alpinetin Modulates Neuroinflammation Pathways
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Alpinetin (CAS. 36052‐37‐6, purity ≥98%) was purchased from Chemstrong Scientific Co., Ltd. Cy3‐labeled goat anti‐rat IgG, Alexa Fluor 555‐tagged donkey antirabbit/mouse IgG, Alexa Fluor 488‐tagged goat antirabbit/mouse IgG, horseradish peroxidase‐labeled secondary antibodies, antifluorescence quenching agent (containing DAPI), and CCK‐8 reagent were purchased from Beyotime. Lipopolysaccharide (LPS) was purchased from Merck. Anti‐COX‐2, anti‐Iba‐1, anti‐CD68, anti‐GFAP, and anti‐GAPDH antibodies were purchased from Abcam. Primary antibodies against iNOS, p‐JAK2, p‐STAT3, STAT3, β‐actin, Bcl‐xL, GFAP, NeuN, GAP43, and MAP2 were obtained from Cell Signaling Technology. Alexa Fluor 488 phalloidin was also from CST. The anti‐CD11b antibody was bought from Biolegend. The anti‐JAK2 antibody was provided by Affinity. WP1066 purchased from GlpBio. Transwell chamber was purchased from Corning (0.4 μm pores). Calcein AM/propidium iodide (PI) was bought from Solarbio. The JC‐1 MMP assay kit and the ROS fluorescence test kit were from Elabscience. The polymerase chain reaction (PCR) primers were synthesized by Servicebio while the kit for RNA extraction came from EZBioscience. The reagents for quantitative real‐time PCR (qPCR) were purchased from Vazyme.
4
Protein Extraction and Western Blotting
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The extraction of total protein from cells was performed employing RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). After the samples were mixed with 5×loading buffer and denatured in boiling water for 5 min, the separation of the proteins was completed via SDS-PAGE, followed by subsequent transfer of separated proteins onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China). After that, the membranes were blocked in 5% skimmed milk for 2 h at room temperature and then incubated with primary antibodies against MAPK1 (1: 5000; ab257525; Abcam, Shanghai, China) and β-actin (1: 2000; ab5694; Abcam, Shanghai, China) overnight at 4 °C. Next, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:1000, Beyotime, Shanghai, China) at room temperature for 2 h. The development of protein bands was conducted employing an enhanced chemiluminescent kit (Biozym, Hessisch Oldendorf, Germany).
5
Quantitative Western Blot Analysis
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The Western blot was implemented as previously described [31 (link)]. After extracting total protein from tissues and cells with Radio-Immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), sodium dodecyl sulfate polyacrylamide gel electrophoresis was implemented, and then electroblot of the protein was onto polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Block of the membrane was with 5% skim milk, and then incubation was with the following primary antibodies: KANSL2 (1:1000, HPA038497, MilliporeSiGBMa) and β-actin (1:1000; ab181602, Abcam). Subsequently, the incubation of the membrane was with horseradish peroxidase-labeled secondary antibodies (1:2000; Beyotime). Visualization of the protein was via exerting an electrochemiluminescence kit (Promega, Madison, Wisconsin, USA).
6
Western Blot Protein Detection Protocol
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Cell lysates were prepared as previously reported.56 (link) For western blotting, whole-cell lysates (20–40 μg per well) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The resolved proteins were transferred to 0.2-μm polyvinylidene difluoride membranes, which were subsequently immersed in Quickblock blocking buffer (Beyotime Biotechnology, China, cat. no. P0233) for 0.5–1 h at room temperature. The membranes were then incubated with the appropriate primary antibodies (Supplementary Table 2 ) overnight at 4 °C before being incubated with the appropriate horseradish peroxidase-labeled secondary antibodies (Beyotime Biotechnology) for 2 h at room temperature. The bands were detected using a BeyoECL Plus Chemiluminescence Detection Kit (Beyotime Biotechnology). Images were acquired using an Amersham Imager 600 (GE Healthcare, Russellville, AR, USA).
7
Protein Expression Analysis by Western Blot
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Cells were lysed on ice for 20 min using RIPA lysis buffer (Beyotime) with protease inhibitor cocktail (Sangon Biotech, China) and the protein lysates were collected. The protein concentration of each sample was determined using a Pierce BCA protein detection kit (Thermo Fisher Scientific). Protein lysates were boiled in 5× protein-loading buffer (Beyotime) at 95 °C for 10 min for denaturing, and then separated by 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were transferred to a polyvinylidene difluoride membrane (Millipore, USA), blocked with 5% skim milk for 1 h, and the membrane was incubated with primary antibodies at 4 °C overnight. The membrane was then washed three times with Tris-buffered saline with Tween 20 (TBST), incubated with horseradish peroxidase-labeled secondary antibodies (Beyotime) at room temperature for 2 h, and washed three times with TBST. The protein bands were then visualized with ECL reaction reagents (Beyotime) using a Western Lightning Gel Imaging System (Tanon 4600; Tianneng Technology Corporation, Shanghai, China). Densitometric analysis of individual blot signals was carried out based on three independent western blot experiments using Image J software.
8
Mitochondrial Dynamics in Renal Cells
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Renal tissue and HK2 cells were homogenized and lysed with T-PER solution containing protease and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice. Mitochondria were isolated from HK2 cells by differential centrifugation utilizing a commercial Mitochondria Isolation Kit (Beyotime, Shanghai, China). Following quantification with a BCA Kit (Beyotime, Shanghai, China), the indicated protein samples (30 μg) were fractionated by 10–12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PVDFs, Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 60 min, the PVDFs were incubated with primary antibodies against rabbit/mouse anti-human/mouse LIGHT, LTβR, Bcl2, Drp1, Bcl2, Parkin, TOMM20, ULK1, and α-SMA (1:500, Abcam, Cambridge, UK), rabbit anti-mouse/human MFF, Mfn1, Mfn2, phosphor-Drp1 Ser616, BNIP3 (1:500, CST, Danvers, MA, USA), rabbit anti-mouse/human HVEM (1:800, Santa Cruz, USA), or rabbit anti-mouse Bak, β-actin (1:800; Beyotime, Shanghai, China) overnight at 4 °C. The next day, the blots were incubated with horseradish peroxidase-labeled secondary antibodies (1:1000, Beyotime, Shanghai, China). The blots were detected utilizing an Enhanced Chemiluminescence Kit (Solarbio, Beijing, China) and quantified with ImageJ software (NIH, Bethesda, MD, USA).
9
Protein Expression Analysis of NPC and Exosomes
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To determine the relative protein expression of CD63, Tsg101, P16, P21 and P53, NPC and SNPC‐Exo were lysed with cell lysis buffer (Beyotime Biotechnology) containing 1% phenylmethylsulfonyl fluoride (Beyotime Biotechnology) and then centrifuged at 14,000 g at 4°C for 5 min to discard the cell debris. NPC lysates and SNPC‐Exo lysates were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked using 5% non‐fat milk in TBST, incubated with specific antibodies overnight at 4°C and then washed thrice with TBST. The membranes were incubated with a horseradish peroxidase‐labeled secondary antibody (Beyotime Biotechnology dilution 1:4000) for 2 h and visualized using an Enhanced Chemiluminescence Substrate (Beyotime Biotechnology) and Image Quant Las4000. The relative expressions of the target protein to glyceraldehyde 3 phosphate dehydrogenase (GAPDH) were calculated using the Image J software. The antibodies involved are as follows: GAPDH (Beyotime 1:1000 dilution), CD63/Tsg101/Calnexin (Santa Cruz 1:200 dilution), and P16/P21/P53 (Protein TECH 1:500 dilution).
10
Immunohistochemical Profiling of Brain Tumor Tissues
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Immunohistochemistry was performed with LAMP-2A (1:100, Abcam, ab18528), N-CoR (1:100, Santa Cruz, sc-1609) for cells and N-CoR (1:100, Novus, CO, US, NBP1-28,863) for tissues. After being equilibrated at − 20 °C for 15 min, brain tumor tissues were sectioned at a range of 5–7 μm and fixed with 4 °C pre-cold acetone. Cultured cells growing on glass slides were fixed with 4% of formaldehyde. The tissue or cell sections were incubated with 3% of H2O2 for 10 min, followed by 10% of BSA as blocking solution for 1 h at room temperature. Then the sections were incubated with the primary antibodies overnight at 4 °C and hybridized with horseradish peroxidase labeled secondary antibody (Beyotime, China) for 1 h at room temperature. The diaminobenzidine kit (DAB kit; Long Island Biotec, Shanghai, China) was applied for visualization and hematoxylin (BASO, China) for counterstain.
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