Tissues were washed with PBS and then homogenized in lysis buffer. The extracted 50 μg of tissue was fractionated on a Bis-Tris gel and transferred to a 0.2-μm nitrocellulose membrane. It was blocked with 5% BSA in Tris-buffer saline with Tween-20 and incubated with a rabbit polyclonal cleaved caspase-3 (1:80; Abcam, Beijing, China) or a rabbit polyclonal Alanine Transaminase (1:1000; Abcam, Beijing, China) overnight. The membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:3000; Abcam, Beijing, China). Bands were detected using a chemiluminescent peroxidase substrate (ECL, Amersham, Beijing, China) and exposed on a radiograph film (Kodak XBT-1, Beijing, China).
Xbt 1
Manufactured by Kodak
Sourced in United States
The XBT-1 is a laboratory equipment product manufactured by Kodak. It is designed for precise temperature control and measurement in a variety of scientific applications. The core function of the XBT-1 is to provide accurate and reliable temperature regulation within a specified range.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Southern Biotech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Horseradish peroxidase labeled goat anti rabbit igg, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Caspase 3, by Abcam (1 mentions)
P mtor ser2448, by Santa Cruz Biotechnology (1 mentions)
P tau, by Abcam (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Beclin 1, by Abcam (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Pmsf and phosphatase inhibitor, by Beyotime (1 mentions)
Bm0627, by Abcam (1 mentions)
Ba1051, by Boster Bio (1 mentions)
Ba1054, by Boster Bio (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
10 protocols using xbt 1
1
Western Blot Analysis of Apoptosis and Liver Enzyme Markers
2
SDS-PAGE Protein Immunoblotting
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Equal amounts of protein (40–50 mg) were size-fractionated using 6–15% SDS-PAGE gradient gels. The resolved proteins were electrophoretically transferred onto polyvinylidene difluoride membranes and analyzed by immunoblotting using an ECL chemiluminescence reagent and XAR film (Kodak, XBT-1) according to the manufacturer's protocol. Primary antibodies were used at optimized dilutions along with the appropriate HRP-conjugated secondary antibodies. The data were collected from at least three independent experiments.
3
Hippocampal Protein Expression Analysis
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Experiments were conducted as previously described.33 The harvested hippocampal tissues were homogenized using lysis buffer supplemented with protease and phosphatase inhibitors on ice. Equal quantities of the proteins (40 μg/well) were separated by SDS‐PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore). The membranes were incubated at 4°C overnight with primary antibodies against the following proteins: mTOR (1:1000, Abcam), p‐mTOR (Ser2448, 1:1000, Santa Cruz Biotechnology, Inc.), p‐Tau (Ser396, 1:500, Abcam), LC3 (1:500, Cell Signaling Technology), Beclin‐1 (1:1000, Abcam), P62 (1:1000, Cell Signaling), cleaved caspase‐3 (1:500, Cell Signaling), Bax (1:1000, Cell Signaling), and Bcl‐2 (Cell Signaling), as well as β‐actin (Cell Signaling), which was used as the internal reference protein. The membranes were then incubated with the corresponding secondary antibodies (1:500, Cell Signaling). After that, the blots were developed with an enhanced chemiluminescence reagent (Pierce) and detected by an X‐ray film (XBT‐1, Eastman Kodak Company). The band intensity was quantified by densitometric analysis using the Image J software (NIH). Relative protein expression levels were obtained by normalizing to β‐actin.
4
Western Blotting Protocol with Modifications
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Western blotting was performed according to the method of Yan et al. [23 (link)] with minor modifications. The cell lysate was prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer. Cell lysates were resolved by electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA; IPVH00010). The blot was then probed with primary antibody followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies. The signal was visualized by enhanced chemiluminescence (Fisher/Pierce, Rockford, IL, USA; 32106) and recorded on an X-ray film (Eastman Kodak Company, Rochester, NY, USA; XBT-1). The whole western blot figures can be found in Figure S2 .
5
Western Blot Protein Analysis
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Western blot analyses were essentially conducted as described previously [34 (link)]. Briefly, ice-cold RIPA (radio-immuno-precipitation assay) buffer containing protease inhibitor was used to lyse the cells. Proteins of the samples were separated by electrophoresis and then transferred to a nitrocellulose membrane (PALL, Pensacola, FL, USA). The membrane was subsequently incubated with primary antibodies following the incubation with secondary antibody. The immunoreacted bands were visualized by enhanced chemiluminescence (Fisher/Pierce, Rockford, IL, USA) and recorded on an X-ray film (Eastman Kodak Company, Rochester, NY, USA; XBT-1).
6
Western Blot Analysis of Protein Signaling
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Total proteins were isolated using RIPA lysis buffer with added PMSF and phosphatase inhibitors (BEYOTIME), and were quantified using a BCA protein assay kit (BEYOTIME). The proteins were separated using 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After being blocked with 5% fat-free milk, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-Periostin (Abcam, ab14041, 1:1000), mouse anti-β-actin (BOSTER, BM0627, 1:200), rabbit anti-ILK (Abcam, ab76468, 1:1,000), rabbit anti-pAkt (CST, 4060, 1:2,000), rabbit anti-Akt (CST, 4691, 1:1,000), rabbit anti-pGSK3β (Abcam, ab68476, 1:1,000), rabbit anti-GSK3β (Proteintech Group, Inc., 22104-1-AP, 1:1,000). Then, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (BOSTER, BA1051, 1:50,000) or HRP-conjugated goat anti-rabbit IgG (BOSTER, BA1054, 1:50,000) for 2 h at 37°C. The blots were visualized with Pierce™ ECL western blotting substrate (Thermo, NCI5079) and then exposed to X-ray films (Kodak, XBT-1). The relative protein levels were quantified using Image J software (NIH, Bethesda, MD, USA).
7
Western Blot Analysis of NRG1 Protein
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Proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Beyotime). The concentrations of proteins were determined with bicinchoninic acid (BCA) kit (Thermo Scientific). Proteins (40 µg) were isolated with 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinyl difluoride (PVDF) membranes (Cat. No. IPVH00010; MILLIPORE). The membranes were blocked with 5% skimmed milk (BD Biosciences) and treated with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1 : 1000, KC-5G5) and NRG1 (Cat. No. ab180808; 1 : 10000, Abcam) overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2000, Southern Biotech, 4050-05) for 2 h and treated with enhanced chemiluminescent reagents (Cat. No. WBKLS0500; MILLIPORE). The signals were examined with medical X-ray films (XBT-1, Eastman Kodak Company, NY, USA).
8
SDS-PAGE and Immunoblotting Protocol
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Equal amounts of protein (40–50 mg) were size-fractionated using 6%–15% SDS-PAGE gradient gels. The resolved proteins were electrophoretically transferred onto polyvinylidene difluoride membranes and analyzed by immunoblotting using an ECL chemiluminescence reagent and XAR film (Kodak, XBT-1) according to the manufacturer’s protocol. Primary antibodies were used at optimized dilutions along with the appropriate HRP-conjugated secondary antibodies. The data were collected from at least three independent experiments.
9
Western Blot Protein Expression Analysis
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Proteins were extracted using RIPA buffer (Beyotime, Shanghai, China). The concentration of proteins was determined by BCA kit (Thermo Scientific). Proteins (40 μg) were isolated by 8% SDS-PAGE, and transferred to polyvinyl fluoride (PVDF) membrane (PMILLIPORE, IPVH00010). The membranes were blocked with 5% nonfat dried milk (BD Biosciences), and then were incubated with primary antibodies overnight at 4 °C. The next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000, Southern Biotech, 4050-05) for 2 h. The membranes were treated with the enhanced chemiluminescent reagents (MILLIPORE, WBKLS0500). The signals were examined by medical X-ray film (XBT-1, Eastman Kodak Company, NY, and USA). The primary antibodies in the present study were glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000, KC-5G5), anti-Bax (1:1000, Abcam; cat no. ab32503), anti-caspase-3 (1:100, Santa Cruz Biotechnology, cat no. sc-7148), anti-tissue inhibitors of metalloproteinase (anti-TIMP; 1:1000, Abcam, ab86482), anti-matrix metallopeptidase 2 (anti-MMP2; 1:1000, Abcam, ab37150), and anti-MMP9 (1:1000, Abcam, ab73734).
10
Protein Extraction and Western Blot Analysis
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Mouse spinal cord tissue and BV‐2 cells were lysed with RIPA buffer (Solarbio) to extract total protein, and the protein concentration was detected using a BCA kit (Beyotime). Equal amounts of protein were separated by 12% SDS‐PAGE and then transferred to PVDF membranes (Millipore). Membranes were then blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies including anti‐rabbit TP53INP2 (DF14312, Affinity Biosciences LTD, dilution rate: 1:1000) anti‐rabbit cleaved‐Caspase3 (9661S; Cell Signaling Technology, dilution rate: 1:1000) and anti‐rabbit GAPDH (5174; Cell Signaling Technology, dilution rate: 1: 1000). The primary antibodies were diluted with 5% skimmed milk. The next day, the membrane was washed three times with TBST buffer. The membranes were then incubated with horseradish peroxidase‐labeled secondary antibodies (AS1107; ASPEN, dilution rate: 1:10,000) for 2 h and visualized with ECL chemiluminescence solution (AS1059; ASPEN) using Kodak medical X‐ray film (XBT‐1; Kodak) in darkroom. AlphaEaseFC software processing system was used to analyze the optical density value of the target band.
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