2,4,6-Trinitrobenzene sulfonic acid solution (TNBS), cyclosporine A (Cys A), Rhodamine 123 (Rho 123), FK506, LY 335979, Stattic, Bay 117082, methanol and acetonitrile were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Human IL-6, IL-1β, TNF-α, IL-17 and LPS were all purchased from Peprotech (Rocky Hill, USA). APC-conjugated anti-mouse CD3, CD14, CD19 antibodies were purchased from Biolegend (San Diego, CA, USA). FITC-conjugated anti-mouse P-gp antibody was purchased from Beijing Biosynthesis Biotechnology Co., Ltd (Beijing, China). FITC-conjugated anti-human P-gp antibody was purchased from BD Bioscience (San Jose, CA, USA). Monoclonal antibodies against stat3, p-stat3, p65, p-p65, lamin B, and β-actin and horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). A SYBR Prime Script RT-PCR Kit was purchased from Takara Bio Inc. (Otsu, Shiga, Japan). Deionized water was prepared using a Milli-Q system (Millipore, Milford, MA, USA) and was used throughout the study.
Horseradish peroxidase conjugated goat anti mouse rabbit igg secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibodies are laboratory reagents used to detect the presence of mouse or rabbit primary antibodies in various immunoassays. The horseradish peroxidase enzyme conjugated to the secondary antibody enables the visualization of bound primary antibodies through a colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Bay11 7082, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Il 17, by Thermo Fisher Scientific (1 mentions)
Ly 335979, by Merck Group (1 mentions)
Cyclosporine a, by Merck Group (1 mentions)
Sybr primescript rt pcr kit, by Takara Bio (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
N methyl n trimethylsilyltrifluoroacetamide mstfa, by Thermo Fisher Scientific (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Nadp nadph quantitation colorimetric kit, by Abcam (1 mentions)
N heptane, by Merck Group (1 mentions)
Methoxamine hydrochloride, by Merck Group (1 mentions)
Pifithrin α pft α, by Selleck Chemicals (1 mentions)
Myristic 1 2 13c2 acid, by Merck Group (1 mentions)
Tanon 5200, by Shanghai Tanon Science & Technology (1 mentions)
Af5216, by Affinity Biosciences (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
4 protocols using horseradish peroxidase conjugated goat anti mouse rabbit igg secondary antibody
1
Immune Modulatory Factors Protocol
2
Myristic Acid Metabolism Modulation
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Myristic-1,2-13C2 acid, methoxamine hydrochloride and menadione (Mena) were purchased from Sigma-Aldrich (USA). 5-13C-glutamine was purchased from Camabridge Isotope Laboratories, Inc. (USA). N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and chlorotrimethylsilane (TMCS) were purchased from Pierce Chemical Co. (USA). Pifithrin-α (PFT-α) was purchased from Selleckchem (USA). HPLC-grade acetonitrile, methanol and n-heptane were purchased from Merck (Germany). Deionized water was prepared by a Milli-Q system (Millipore, USA). N-acetyl cysteine (NAC), glutathione (GSH) and an oxidized glutathione (GSSG) Test Kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). NADP/NADPH Quantitation Colorimetric Kit was purchased from BioVision, Inc. (USA). The primers for glucose-6-phosphate dehydrogenase (G6PD), mdr1, p53 and β-actin for qPCR analysis were synthesized by Invitrogen (Life Technologies, USA). Monoclonal antibodies against Chk2, p-Chk2, p53, p65, histone, and horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibodies were purchased from Cell Signalling Technology (Danvers, MA, USA). The antibody for GAPDH was purchased from Bioworld Technology (Dublin, Ohio, USA).
3
Immunoblotting of Cell Signaling Proteins
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Lysates were harvested from cells in RIPA lysis buffer (P0013B, Beyotime) containing a protease inhibitor cocktail. Equal amounts of protein were separated via sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes electrophoretically. After incubation with blocking buffer, the membrane was incubated with antibodies against iNOS (1:1000, 18985‐1‐AP, Proteintech group), GFAP (1:5000, 60190‐1‐Ig, Proteintech group), ZO‐1 (1:1000, 21773‐1‐AP, Proteintech group), Occludin (1:1000, 27260‐1‐AP, Proteintech group), Claudin‐5 (1:1000, AF5216, Affinity), TAF1 (1:1000, 20260‐1‐AP, Proteintech group), Histone‐H3 (1:1000, 17168‐1‐AP, Proteintech group), or GADPH (1:3000, 60004‐1‐AP, Proteintech group) overnight at 4°C. After three washes, the membrane was incubated with a horseradish peroxidase‐conjugated goat anti‐mouse/rabbit IgG secondary antibody (1:2000, 7076P2/7074P2, Cell Signaling). Signals were detected by Automatic Chemiluminescence Image Analysis System (Tanon 5200, Tanon Science & Technology). Quantification of the individual protein bands was performed via densitometry using Image J software.
4
Protein Expression Analysis Protocol
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Proteins were extracted in RIPA lysis buffer (Beyotime, Shanghai, China), separated on sodium dodecyl sulfate polyacrylamide gels (8% and 12%) and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20, probed with antibodies recognizing p-ERK/ERK (1:1,000, Cell Signaling, #9101S/#9107S), p-JNK(1:1,000, Santa Cruz, sc-6254)/JNK(1:1,000, Cell Signaling, #9252S), p-p38/p38(1:1,000, Cell Signaling, #9211S/#9212S), p-AKT/AKT (1:1,000, Cell Signaling, #9271S/#9272S), NF-κB p65 (1:1,000, Cell Signaling, #3033S), STAT3 (1:1,000, Cell Signaling, #12640S), histone H3 (1:1,000, Cell Signaling, #9715S), claudin-5 (1:1,000, Abcam, ab15106), occludin (1:1,000, Life Technology, #33–1500), ZO-1 (1:1,000, Life Technology, #402300), p53 (1:1,000, Santa Cruz, sc-6243), PUMA (1:1,000, Santa Cruz, sc-28226) and β-actin (1:1,000, Bioworld, BS6007M) overnight at 4 °C, and then incubated with a horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody (1:2,000, Cell Signaling, #7076P2/#7074P2). A Microchemi 4.2® (DNR, Israel) digital image scanner was used for detection, and the band intensity was quantified using ImageJ software (NIH).
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