Proteins were resolved in 12% SDS-PAGE, transferred to PVDF membranes (GE), and incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA), GAPDH (30201ES20, Yeasen, China), mCherry (ab125096, Abcam, UK), α-Tublin (11224-1-AP, Proteintech, USA), his (30404ES60, Yeasen, China). Second antibodies are peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) (33101ES60, Yeasen, China) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (33201ES60, Yeasen, China).
Peroxidase conjugated goat anti rabbit igg h l
Manufactured by Yeasen
Sourced in China
Peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) is a secondary antibody conjugated with horseradish peroxidase enzyme. It is designed to detect and bind to rabbit IgG antibodies, both heavy and light chains.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase affinipure goat anti mouse igg h l, by Yeasen (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab81299, by Abcam (2 mentions)
Ripa lysis buffer, by Yeasen (2 mentions)
α tublin, by Proteintech (1 mentions)
Gapdh, by Yeasen (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Ac026, by ABclonal (1 mentions)
T40044, by Abmart (1 mentions)
Goat anti mouse igg h l, by Yeasen (1 mentions)
T40056, by Abmart (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ecl reagent, by Yeasen (1 mentions)
Ab207612, by Abcam (1 mentions)
Anti nampt antibody, by Abcam (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Ab6160, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
Bca protein quantification kit, by Yeasen (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
7 protocols using peroxidase conjugated goat anti rabbit igg h l
1
Western Blot Analysis of Protein Expression
2
Cerebral Cortex Protein Analysis
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Total proteins were extracted from the right cerebral cortex in the ischemic penumbra and quantitated using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated with 8–12% SDS-PAGE gels and electrically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies against Bcl-2 (1:1000, T40056, Abmart, Shanghai, China), Beclin1 (1:2000, ab207612, Abcam, Cambridge, UK), Bax (1:10,000, 50599-2-Ig, Proteintech, Wuhan, China), caspase3 (1:1000, T40044, Abmart, Shanghai, China), LC3B (1:2000, T40044, Abcam, Cambridge, UK), ZO-1(1:2000, 21773-1-AP, Proteintech, Wuhan, China), Occludin (1:10,000, 66378-1-Ig, Proteintech, Wuhan, China), β-actin (1:100,000, AC026, Abclonal, Wuhan, China), and GAPDH (1:10,000, 60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. They were then incubated in Peroxidase-Conjugated Goat Anti-Rabbit IgG(H+L) or Goat Anti-Mouse IgG(H+L) (Yeasen, Shanghai, China) diluted at 1:10,000 for 1 h at room temperature. After chemiluminescence detection had been performed using ECL reagents (Yeasen, Shanghai, China) and a Fusion Edge Multi-function Imaging System (Vilber, Collégien, France), the relative optical densities of the protein bands were analyzed using ImageJ software.
3
Western Blot Analysis of NAMPT Protein
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Cells were lysed and equivalent amounts of protein samples (25 μg per sample) were run on 10% SDS-PAGE gels at 60–120V constant voltage to separate them and transferred onto Immobilon-P PVDF membranes (Millipore) at a constant current (200mA for 1h). Membranes were blocked using Blocking Buffer for Wb (Beyotime, cat#P0252) for 2 h at room temperature. Membranes probed for NAMPT and GAPDH were reprobed with the Anti-NAMPT antibody (Abcam, cat#ab236873, EPR21984, 1:1000 diluted) and GAPDH Rabbit pAb (Yeasen, cat# 30202ES40, 1:3000 diluted) overnight at 4°C. Next, they were washed three times with TBS-T, followed by incubation with Peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) (Yeasen, cat#33101ES60, 1:5000 diluted) for 1 h at room temperature. Following washing with TBS-T. Densitometer and ImageJ software immunoreactivity signals were detected by enhanced chemiluminescence (Tanon 5200, Shanghai, China).
4
Protein Quantification and Western Blot Analysis
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Protein was extracted from cells, liver, and kidney using RIPA lysis buffer (Yeasen). Protein extracts were quantified by the BCA Protein Quantification Kit (Yeasen). For γH2AX, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to separate the protein bands, and then, proteins were transferred from gel to the PVDF membrane, blocked with 5% milk powder in TBST for 1 hour. Membranes were incubated with specific antibodies for γH2AX (ab81299, Abcam) and tubulin (ab6160, Abcam). For collagen, 8% SDS-PAGE was run. Membranes were incubated with specific antibodies for GAPDH (60004-1-lg, Proteintech) and Collagen I (ab260043, Abcam). Then, immunocomplexes were detected by Peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) (33101ES60, Yeasen), Peroxidase-Conjugated Goat Anti-Rat IgG (H+L) (33301ES60), and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (33201ES60, Yeasen) followed by enhanced chemoluminescence (New Cell & Molecular Biotech). The membrane was finally photographed by imaging techniques (Tanon-4600SF). ImageJ was used to quantify the intensity.
5
Western Blot Analysis of Insect OBPs
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Antennae from M. alternatus and D. helophoroides in different treatments (non-injected, dsGFP-injected, and dsMaltOBP24-injected or dsDhelOBP10-injected) were homogenized in RIPA Lysis Buffer (Beyotime) to obtain total proteins. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime). 20 μg of the samples were denatured at 100 °C for 5 min in 4X SDS-PAGE loading buffer, separated by 15% SDS-PAGE gels and transferred to 0.45 μm PVDF membranes (Millipore, USA). After being blocked for 2 h at room temperature, the membranes were washed and incubated with specific primary antibodies diluted in 5% milk/PBST at 1:1500. The primary antibodies used in this study were as follows: Rabbit anti-MaltOBP24 antibody, Rabbit anti-DhelOBP10 antibody, Rabbit β-Tubulin Antibody (Yeasen), and Rabbit GAPDH Antibody (AtaGenix). Peroxidase-Conjugated Goat Anti-Rabbit IgG (H + L) (Yeasen) diluted in 5% milk/PBST at 1:4000 was used as the secondary antibody. Electrochemiluminescence (ECL) (Beyotime) and the chemiluminescence imaging system with a charge-coupled device (CCD) camera (Bio-Rad) was used for target protein detection.
6
Western Blot Analysis of Cellular Proteins
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Cells were lysed in ice-cold RIPA Lysis and Extraction Buffer (Thermo, United States) supplemented with protease (Roche, Switzerland) and phosphatase inhibitor cocktail (Roche). The protein concentrations were determined using BCA Protein Assay Kit (Pierce, United States). Protein lysate was separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis system (SDS-PAGE) and transferred onto PVDF membranes (Millipore, United States). The membrane was incubated in blocking buffer (1 × TBST with 5% BSA) for 2 h at room temperature and then incubated in diluted primary antibodies: anti-CPSF6 (1:1,000; Abcam, United Kingdom), anti-VHL (1:1,000, Sangon), anti-GAPDH (1:1,000; Proteintech, China), anti-β-actin (1:5,000; Sigma, United States), and anti-β-tubulin (1:1,000; Yeasen, China), and secondary antibody: peroxidase-conjugated goat anti-rabbit IgG (H+L) (1:5,000; Yeasen). The membrane was visualized using ECL start Western Blotting Substrate (GE Healthcare Life Sciences, United States). The intensities of the bands were qualified by ImageJ (National Institutes of Health, United States).
7
Western Blot Analysis of Liver Proteins
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Mice liver was homogenated in RIPA lysis buffer (strong, Yeason) added with General Protease Inhibitor Cocktail (Absin) and PMSF (Tansoole), and desaturated using 3 × SDS loading buffer (CST). Protein extracts were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filter membrane, and blocked with 5% milk powder in 0.02% Tween in TBS for 1 h. Membranes were incubated with specific monoclonal antibodies for P21 (sc-6246, Santa Cruz), γ-H2AX (ab81299, Abcam), and GAPDH (60004-1-lg, Proteintech) overnight at 4 °C. Immunocomplexes were visualized using Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (33201ES60, Yeason) and peroxidase-conjugated goat anti-rabbit IgG (H + L) (33101ES60, Yeason), followed by enhanced chemoluminescence (New Cell & Molecular Biotech). Quantification of the grey value was done using ImageJ.
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