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4 protocols using horseradish peroxidase hrp conjugated goat anti mouse igg

1

Sciatic Nerve Injury Protein Analysis

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Sciatic nerves and left L3-L5 DRGs were dissected and homogenized rapidly on ice at the time point of one week and two weeks after sciatic injury. The protein concentration of each sample was determined by Bradford assay (Sangon Biotech, China). Twenty micrograms homogenate of DRGs and 30 μg homogenate of sciatic nerves were uploaded onto 10% acrylamide gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was then transferred onto polyvinylidene fluoride membranes. Afterward, the membranes were blocked with 5% no-fat milk solution and then incubated with mouse anti-TRPV1 (1:4000, Biosensis, USA), mouse anti-GFAP (1:2500, Novus Biologicals, USA), or rabbit anti-GAP-43 (1:5000, Bioworld, USA) overnight at 4°C. On the second day, the membranes were washed with 0.1% Tris-buffered saline and Tween 20, 3× 5 min and then incubated for 1 h in horseradish peroxidase (HRP)-conjugated goat antimouse IgG (1:5000, Sangon Biotech, China) or HRP-conjugated goat antirabbit IgG (1:5000, Sangon Biotech, China). Finally, the protein bands were detected after incubated in Easy See Western Blot ECL reagent (Sangon Biotech, China) and exposed onto X-ray film in dark room. Meanwhile, rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Bioworld, USA) was used as a loading control.
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2

SDS-PAGE and Immunoblot Analysis

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SDS-PAGE was performed with 12% polyacrylamide gel using a Tris-glycine or Tris-tricine buffer system. Protein samples were precipitated with 20% (wt/vol) trichloroacetic acid (TCA) and washed with acetone before being subjected to SDS-PAGE analysis. In some cases, 8 M urea was included in the loading buffer and the gel for urea-SDS-PAGE. The anti-His tag monoclonal antibody (1:10,000) (Novagen) and the alkaline phosphatase (AP)-conjugated goat anti-mouse IgG secondary antibody (1:5,000) (Abbkine, China) were used for immunoblot analysis, and the immunoreactive proteins were visualized using the BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium) chromogen kit (Solarbio, Beijing, China) as described previously (64 (link)). In some cases, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was used as the secondary antibody (1:5,000) (Sangon Biotech, China), and the signals were detected using Pierce ECL Western blotting substrate (Thermo Fisher Scientific, USA). The images were photographed using the GE Amersham Imager AI680 with an exposure time of 1 or 5 min.
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3

Immunoassay Development for Organochlorine Pesticides

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Endosulfan, toxaphene, chlordane, heptachlor, hypoxanthine aminopterin, bovine serum albumin (BSA), ovalbumin (OVA), carbodiimide (EDC), Freund’s complete adjuvant, Freund’s incomplete adjuvant, chloroauric acid (HAuCl3·4H2O), trisodium citrate, thymidine medium (HAT) and hypoxanthine-thymidine medium (HT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, 3,3′,5,5′-tetramethylbenzidine (TMB) and Freund’s complete and incomplete adjuvant were acquired from Sangon Biotech Co., Ltd. (Shanghai, China). Immunogen and coating antigen were prepared in our laboratory. All other reagents were acquired from the National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China).
The microplate reader (1201–7044) was obtained from Thermo Fisher Scientific (Shanghai, China) Co., Ltd. (USA). The CT300 CNC strip cutting machine, HM3030 XYZ three-dimensional dispensing platform, ZQ3500 CNC fast chopping machine were obtained from shanghai Kinbio Tech Co., Ltd. (Shanghai, China). The gas chromatography-mass spectrometer (GC-MS) was obtained from Thermo Fischer Scientific (USA).
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Recombinant Plasmid pPIC9K::FIP-glu-His Expression in Pichia pastoris

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The recombinant plasmid pPIC9K::FIP-glu-His and Pichia pastoris GS115 strain were preserved in our laboratory. HisSep nickel-nitrilo-triacetic acid (Ni-NTA) agarose resin was purchased from Yeasen (Shanghai, China). The restriction enzyme Sac I was purchased from Takara (Beijing, China). DPPH and vitamin C were purchased from Innochem (Beijing, China). The CCK-8 kit and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and the Bradford protein assay kit were procured from Sangon (Shanghai, China). Other reagents were of analytical grade.
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