Western blot analyses were performed to evaluate target protein expression. Briefly, protein lysates (~20 μg per sample) were loaded and run on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to NC membranes (Millipore Corporation, MA, USA), and blocked with 5.0% nonfat milk for 3 h. The membranes were incubated with primary antibodies overnight at 4°C and then washed with Tris-buffered saline Tween-20 (TBST) three times for 10 min. Afterwards, membranes were incubated with the secondary antibodies for 2 h at room temperature and washed again. The bands on the membranes were visualized using an ECL system (Beyotime Institute of Biotechnology). Finally, membranes were exposed with an Odyssey infrared imaging system (LI-COR Biosciences, NE, USA). The following antibodies were used: anti-human FOLR1 (1:5000 dilution; Abcam, Cambridge, UK; CAT# ab221543), anti-human PCFT (1:300 dilution; Santa Cruz Biotechnology, Dallas, TX; CAT# sc-393460), anti-human RFC (1:300 dilution; Santa Cruz Biotechnology; CAT# sc-390948), anti-human GAPDH (1:5000 dilution; Abcam; CAT# ab181602), and HRP-labeled secondary antibodies (1:5000 dilution; Proteintech Group, Rosemont, IL).
Hrp labeled secondary antibody
Manufactured by Proteintech
Sourced in China, United States
The HRP-labeled secondary antibody is a laboratory reagent used for the detection and visualization of target proteins in various immunoassay techniques, such as Western blotting and ELISA. It consists of a secondary antibody conjugated with the enzyme Horseradish Peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction to produce a signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Protease and phosphatase inhibitor, by Beyotime (2 mentions)
Enhanced chemiluminescence system, by Merck Group (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Bcl 2, by Proteintech (2 mentions)
β actin, by Proteintech (2 mentions)
Nc membrane, by Merck Group (1 mentions)
Ecl system, by Beyotime (1 mentions)
Ab181602, by Proteintech (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab5675, by Merck Group (1 mentions)
Bca kit, by Solarbio (1 mentions)
Nitrocellulose filter nc membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Nc membrane, by GE Healthcare (1 mentions)
Anti vegfr2, by Santa Cruz Biotechnology (1 mentions)
Anti ezh2, by Proteintech (1 mentions)
Anti vegfa, by Proteintech (1 mentions)
25 protocols using hrp labeled secondary antibody
1
Quantitative Analysis of Folate Transporters
2
Hippocampal and Cortical Synaptosome Isolation
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Synaptosomes were isolated from the whole hippocampus or cortex of the brain for analysis of protein levels. Briefly, the whole hippocampus or cortex was homogenized in 10 volumes of Hepes-buffered sucrose [0.32 M sucrose and 4 mM Hepes (pH 7.4)]. The homogenate was centrifuged at 1200g for 10 min to remove cell debris, and the supernatant was centrifuged at 15,000g for 20 min at 4°C. The pellets, containing synaptosomes, were gently resuspended in radioimmunoprecipitation assay buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na3VO4, and leupeptin (0.5 μg/ml)] for further protein analysis. Anti-mGluR5 (AB5675, MilliporeSigma), anti-GluNR2A (19953-1-AP, Proteintech), anti-GluNR2B (21920-1-AP, Proteintech), and anti-PSD95 (20665-1-AP, Proteintech) were used as primary antibodies at a dilution of 1:1000. HRP-labeled secondary antibodies (Proteintech) were used at a dilution of 1:5000. Antibodies against tubulin (10068-1-AP, Proteintech) were used as loading controls. All Western blot quantifications were performed using ImageJ.
3
Western Blot Analysis of Glioma Proteins
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RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Beyotime, Shanghai, China) was used to separate total protein from human glioma cells and tissues. The concentration of total protein was determined with a BCA kit (Solarbio, Beijing, China). An equal amount of total protein (30 µg) was electrophoresed on 10%−12% SDS-PAGE, and then electro-transferred to a nitrocellulose filter (NC) membrane (Millipore, MA, USA). Then, the membranes were blocked with 5% non-fat dry milk in TBST at RT for 1 h. After washing with TBST, the membranes were incubated with subsequent primary antibodies at 4 °C overnight. On the second day, the membranes were incubated with HRP-labeled secondary antibodies (Proteintech, Wuhan, China) at RT for 1 h. An enhanced chemiluminescence system (Millipore, MA, USA) was used to measure protein expression value. The relative quantity of proteins was analyzed by Image J software. The antibodies are shown in Table S2 .
4
Western Blot Analysis of Glioma Signaling
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The glioma cells were lysed using RIPA buffer with Protease and Phosphatase inhibitor (Beyotime). The total protein concentration was examined using a BCA kit. Equal amounts of total protein (30 μg) were electrophoresed in 10%–12% SDS‐PAGE and electro‐transferred onto NC membrane (GE). After blocking with 5% non‐fat dry milk in TBST for 1 h at room temperature (RT), the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with HRP‐labeled secondary antibodies (Proteintech) for 1 h at RT. An enhanced chemiluminescence reagent (Millipore) was used to detect protein expression value. The relative quantity of proteins was analyzed using the Image J software. The primary antibodies used are as follows: anti‐EZH2 (1:2000, #66476‐1‐Ig, Proteintech), anti‐VEGFA (1:1000, #66828‐1‐Ig, Proteintech), anti‐VEGFR2 (1:200, #sc‐6251, Santa‐Cruz Technology), anti‐PhosphoTyr1175‐VEGFR2 (1:1000, #2478, CST), anti‐AKT (1:1000, #9272, CST), anti‐ PhosphoThr308‐AKT (1:1000, #13038, CST), anti‐ERK1/2 (1:1000, #4695, CST), anti‐PhosphoThr202/Tyr204‐ERK1/2 (1:1000, #4370, CST), anti‐GAPDH (1:5000, #60004‐1‐Ig, Proteintech), and anti‐β‐tubulin (1:5000, #10094‐1‐AP, Proteintech).
5
Western Blot Protein Quantification Protocol
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Proteins were extracted from cells and tissues by RIPA solution, which was supplemented with protease inhibitors purchased from Beyotime (China). To determine the protein concentration, a BCA kit from Invitrogen (CA, USA) was utilized. Electrophoresis of an equal amount of total protein (30 µg) was carried out on 10% SDS-PAGE. Subsequently, the protein was electro-transferred onto a nitrocellulose filter (NC) membrane obtained from GE (USA). The membranes were then subjected to blocking with 5% non-fat dry milk in TBST at room temperature (RT) for a duration of 1 h. Following this, primary antibodies were added to the membranes and they were incubated overnight at 4 °C. On the following day, HRP-labeled secondary antibodies purchased from Proteintech (China) were incubated with the membranes at RT for 1 h. Protein expression signal was examined using an enhanced chemiluminescence system from Millipore (MA, USA) and was quantified using Image J software.
6
Immunohistochemical analysis of GSDMD and p-p65 in brain sections
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Brain paraffin sections (4 μm) were hydrated and Tris/EDTA buffer performed heat-mediated antigen retrieval for 20 min. The sections were then processed with 3% H2O2 for 10 min. The sections blocked with 5% BSA for 1 h were incubated with primary antibodies GSDMD (Abclonal), phosphorylated p65 (Abclonal) overnight at 4°C, and then incubated in HRP-labeled secondary antibodies (Proteintech). DAB (Servicebio) was utilized for dyeing while hematoxylin was used for nuclei staining. Images were acquired using the Olympus BX53 microscope (Olympus). The distribution and intensity of GSDMD and p-p65 staining was described by a semiquantitative score in a blinded fashion (0-negative, 1-weak, 2-moderate, 3-strong, and 4-strong and widely distributed) (n = 6/group) (Xu et al., 2020 (link)).
7
Autophagy-Related Protein Expression
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The expression of AMPK, mTOR, unc-51 like autophagy activating kinase 1 (ULK1), BECLin-1, light chain 3 II (LC3-II)/light chain 3 I (LC3-I), and Sequestosome1 (p62) were measured using western blot according to the manufacturer's instructions. After treatment of kidney and lung tissue, the cells were washed three times with cold PBS and lysed. Total cytoplasmic and nuclear protein was collected using cell lysis buffer. The protein concentration was measured using a BCA assay. Equal amounts of protein (25 μg) were separated by 10% SDS-PAGE, followed by transfer to PVDF membranes. The membranes were blocked in 5% BSA at room temperature for 1 h and 1×TBST for 5 min, and then incubated with primary antibodies against AMPK (1:1,000), mTOR (1:1,000), ULK1 (1:1,000), BECLin-1 (1:500), LC3-II/LC3-I (1:500), p62 (1:500), and GAPDH (1:10,000) at 4°C overnight. Anti-GAPDH antibody was selected as an internal reference. Subsequently, the membranes were washed with TBST and incubated with HRP-labeled secondary antibodies (1:5000, Proteintech) at the room temperature for 2 h. Finally, the blots were visualized using ECL (Affinity Biosciences) and the results were analyzed using ImageJ software (National Institutes of Health, USA). The catalog numbers: anti-ULK1 (A00584-1), anti-BECLin-1 (PB9076), anti-LC3-II/LC3-I (BM4827), anti-p62 (PB0458), anti-AMPK (ab32047), and anti-mTOR (ab32028).
8
EBOV GP-specific Antibody ELISA
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EBOV GP-specific binding antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) as described previously45 (link). In brief, 96-well flat-bottom plates were coated with 100 µl purified EBOV GP at 1 µg/ml in PBS at 4 °C overnight. After washing and blocking, serum samples were serially diluted in twofold increments and added in duplicate. After incubation at room temperature for 2 h, the plates were washed, and HRP-labeled secondary antibody was added (Proteintech, IL, USA). After incubation for another 1 h at room temperature, the plates were washed and developed with TMB/E substrate (Merck Millipore, MA, USA). Finally, the reaction was stopped, and the OD450 values were read. The ELISA titers were calculated as a reciprocal endpoint. A negative serum control was run each time the assay was performed. A cutoff value for a positive result was calculated as the mean optical density (at a 1:100 dilution) for the negative sera plus 3 standard deviations (SDs).
9
Western Blot Quantification of Inflammatory Proteins
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The extracted total protein samples were subjected to SDS-PAGE gel electrophoresis and the membranes were transferred to NC membranes by wet transfer method (GE/General Electric, Cat: 1060002) and rapid closure solution (EpiZyme, Cat: PS108P) for 30 min, and the membranes were washed with membrane washing buffer (TBST) three times for 10 min each time. Primary antibodies IL-6, IL-1 , NLRP-3, NRF2, HO-1 (1:1000) and GAPDH (1:2000) were added, and the membranes were incubated at 4 °C overnight. The membrane was washed three times with TBST for 10 min each time and HRP-labeled secondary antibody (1:5000) (Proteintech) was added. The membranes were incubated at 37 °C for 2 h and the relative expression of proteins was analyzed by Image J image analysis system.
10
Western Blot Analysis of TBK1, IRF3, and LC3
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Total protein was isolated using RIPA Lysis Buffer (Beyotime). Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred onto PVDF membranes (Millipore Corporation, Billerica, MA) and probed with rabbit monoclonal anti-TBK1/NAK (D1B4) antibody (3504), rabbit monoclonal anti-phospho-TBK1/NAK (Ser172) (D52C2) antibody (5483), rabbit monoclonal anti-IRF-3 (D83B9) antibody (4302), rabbit monoclonal anti-phospho-IRF-3 (Ser396) (D6O1M) antibody (29047) (Cell Signaling Technology, Boston, Mass, USA), and mouse anti-Map-LC3 antibody (sc-376404) (Santa Cruz, CA, USA). This was followed by HRP-labeled secondary antibody (Protein Tech, Wuhan, Hubei, China). Bands were developed with ECL substrate, visualized using BIO-RAD imaging system and analyzed by Image J analysis software.
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