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Horseradish peroxidase conjugated igg

Manufactured by Cell Signaling Technology
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Horseradish peroxidase-conjugated IgG is a laboratory reagent that consists of horseradish peroxidase enzyme covalently linked to immunoglobulin G (IgG) molecules. The primary function of this product is to serve as a detection and visualization tool in various immunoassay techniques, such as enzyme-linked immunosorbent assays (ELISAs) and Western blotting.

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Lab products found in correlation

Ecl kit, by Thermo Fisher Scientific (3 mentions) Ecl western blotting detection system, by Merck Group (2 mentions) Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Bioworld Technology (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Ecl chemiluminescence system, by Cell Signaling Technology (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) M mlv reverse transcriptase, by Takara Bio (1 mentions) Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions) Phosphatase inhibitor cocktail, by Merck Group (1 mentions) Lysis buffer, by Cell Signaling Technology (1 mentions) D6b6s, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Bradford protein assay, by Bio-Rad (1 mentions) Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions) α sma, by Abcam (1 mentions) Hrp conjugated anti mouse and anti rabbit igg, by Cell Signaling Technology (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions)

10 protocols using horseradish peroxidase conjugated igg

1

Quantification of CSE Expression in EPCs

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Total RNA was extracted using TRIzol reagent (Invitrogen). Double-stranded DNA was synthesised using M-MLV Reverse Transcriptase (TaKaRa, Kusatsu, Shiga, Japan). RT-PCR was carried out using Light Cycler 480 SYBR Green I Master Mix (Roche Diagnostics, Risch-Rotkreuz, Switzerland) in a Light Cycler 480 System. The cycling protocol used was as follows: 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 20 s. The CSE and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers used were seen in Table S2.
EPCs protein was harvested by cell lysis buffer (Cell Signaling Tech) in line with the manufacturer’s instructions. Protein (50 µg) was subjected to sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The following antibody was used CSE (1:1,000, Abnova). Proteins were visualised with horseradish peroxidase-conjugated IgG (1:5,000, Cell Signaling Tech), followed by using of the ECL chemiluminescence system (Cell signaling Tech).
Hu Q., Zhang B., Liu Y., Guo Y., Zhang T., Nie R., Ke X, & Dong X. (2020). The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells. Annals of Translational Medicine, 8(20), 1318.
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2

Quantification of Inducible Nitric Oxide Synthase

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RAW 264.7 cells were seeded in 100 mm dishes. After 24 h treatments, cells were scraped into centrifuge tubes and washed in cold phosphate-buffered saline (PBS) and centrifuged to get pellets. The cell pellets were then lysed using lysis buffer (Cell Signaling, Danvers, MA) containing phosphatase inhibitor cocktails (Sigma Chemical, St. Louis, MO). Protein concentration was determined using a Bradford Protein assay (Bio-Rad). Aliquots containing 20 μg protein were loaded onto a 10% sodium dodecyl sulfatepolyacrylamide gel, transblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), blocked with Tris-buffered saline with 1% Casein with 0.1% Tween-20, and then incubated with each of the primary antibodies of iNOS (D6B6S, #13120, Cell signaling, Danvers, MA) and β-actin (#4970, Cell signaling, Danvers, MA). The membrane was then incubated with horseradish peroxidase conjugated IgG (Cell signaling, Danvers, MA). The bound complexes were detected with Super Signal Chemiluminescent Subtract (Thermo Scientific, Rockford, IL). The immunoblot bands were quantified by densitometry analysis using Image J, and the ratio to β-actin was calculated and presented, setting the value of control as 1.
Zhang S., Zhao Y., Ohland C., Jobin C, & Sang S. (2018). Microbiota facilitates the formation of the aminated metabolite of green tea polyphenol (−)-epigallocatechin-3-gallate which trap deleterious reactive endogenous metabolites. Free radical biology & medicine, 131, 332-344.
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3

Western Blot Analysis of Lung Tissues

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Samples of lung tissues and LA-4 cells were homogenized in RIPA buffer (Cell Signaling, Boston, MA, USA) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein samples in BAL fluid were extracted with the trichloroacetic acid (TCA) precipitation method [29 (link)]. Then, 30 μg of total protein was separated by gel electrophoresis and transferred to a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBST (20 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.05% Tween-20) for 1 h, followed by incubation with the appropriate primary antibodies overnight at 4 °C. Antibodies against SP-A (#BS-10265R, 1:200) and GAPDH (1:500) were obtained from ThermoFisher Scientific (Waltham, MA, USA). Antibodies against E-cadherin (1:500) and α-SMA (1:500) purchased from Abcam (Cambridge, MA, USA). After the membranes were washed with TBST, they were incubated with horseradish peroxidase-conjugated IgG. HRP-conjugated anti-rabbit and anti-mouse IgG were obtained from Cell Signaling Technology, and anti-goat IgG-HRP antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The signals were visualized with enhanced chemiluminescence reagents (Clarity Western ECL Substrate, Bio-Rad, Hercules, CA, USA). The results were analyzed using the ImageJ densitometry system.
Kim K., Shin D., Lee G, & Bae H. (2022). Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis. International Journal of Molecular Sciences, 23(10), 5292.
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4

Quantification of Colonic Protein Expression

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Total protein of colonic tissues and cells was extracted using a protein extraction reagent (Pierce Biotechnology, Rockford, IL, USA). Homogenates containing 20 μg of total protein were separated by SDS-PAGE and transferred to nitrocellulose membranes, and blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20. The membranes were probed with primary antibodies for 12 h, followed by the secondary antibodies horseradish peroxidase-conjugated IgG (Cell Signaling Technology, Beverly, MA, USA) for 1 h. Antibodies against iNOS (ab3523), COX-2 (ab15191), IL-1β (ab9722), TNF-α (ab9739), IκBα (ab32518), p-IκBα (ab133462), p-p65 (ab86299), Lamin B1 (ab16048), NQO1 (ab34173), HO-1 (ab13248), Nrf2 (ab92946) and β-actin (ab8227; Abcam, Cambridge, United Kingdom) were used. The membrane was incubated for 2 h at 25 °C after treatment with the secondary antibodies. After washing with Tris-buffered saline with Tween buffer, the membranes were treated with enhanced chemiluminescence solution and imaged using a Chem-iDoc system (Bio-Rad, Hercules, CA, USA). Imaged bands were quantified with β-actin using ImageJ software (Ver.1.8, National Institute of Health, Sacaton, Arizona, USA).
Bae U.J., Jang H.N., Lee S.H., Kim J.Y, & Kim G.C. (2022). Oenanthe javanica Ethanolic Extract Alleviates Inflammation and Modifies Gut Microbiota in Mice with DSS-Induced Colitis. Antioxidants, 11(12), 2429.
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5

Western Blot Analysis of NLRP3, Caspase-1, and Sirt-1

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Protein was extracted from brain tissue with radioimmunoprecipitation assay lysis buffer (Dallas, TX, USA), and a total of 30 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was then transferred to a nitrocellulose membrane and blocked in 10% skimmed milk at room temperature for 1 h. The membranes were incubated overnight with primary antibodies against NLRP3 (1:500; Invitrogen, Grand Island, NY, USA), cleaved caspase-1 (1:500, Abcam, Cambridge, UK), Sirt-1 (1:300, Cell Signaling Technology, Danvers, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000; Bioworld Technology, St Louis Park, MN, USA) at 4 °C. The membranes were then washed with PBS and incubated with horseradish peroxidase-conjugated IgG (1:5,000, Cell Signaling Technology, Danvers, MA, USA) secondary antibodies at room temperature for 1 h. The protein bands were visualized using an ECL Western Blotting Detection System (Millipore, Billerica, MA, USA) and normalized for GAPDH expression.
Wang J., Guo M., Ma R., Wu M, & Zhang Y. (2020). Tetrandrine alleviates cerebral ischemia/reperfusion injury by suppressing NLRP3 inflammasome activation via Sirt-1. PeerJ, 8, e9042.
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6

Western Blot Analysis of Ischemic Cortex Proteins

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Protein in the ischemic cortex tissue was extracted with a commercial protein extraction kit (Beyotime), and the protein quantity was measured with a BCA protein assay kit (Beyotime), following the manufacturer's protocols.
A total of 40 μg protein samples were separated through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10–15%), and the protein was transferred to nitrocellulose membranes. After the samples were blocked in 10% skimmed milk for 1 h at room temperature, they were incubated with primary antibodies against LC3 (1 : 1000; Sigma-Aldrich), p62 (1 : 1000; Abcam), p-AMPK (1 : 1000; Cell Signaling Technology, MA, USA), AMPK (1 : 1000; CST), Sirt1 (1 : 1000; CST), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 3,000; Bioworld Technology, St Louis Park, USA) at 4°C overnight. The membranes were washed with PBS 3 times, for 5 min each time, and incubated with horseradish peroxidase-conjugated IgG (1 : 5,000, Cell Signaling Technology) secondary antibodies for 2 h at room temperature. Specific protein signals were visualized with an ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). The bands were analyzed by ImageJ software and normalized for GAPDH expression.
Liang H., Chang X., Xia R., Wu W., Guo H, & Yang M. (2022). Magnoflorine Attenuates Cerebral Ischemia-Induced Neuronal Injury via Autophagy/Sirt1/AMPK Signaling Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 2131561.
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7

Kidney and Cell Protein Extraction and Western Blot Analysis

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Total protein from kidneys and cells were extracted as previously described11 (link). Incubation was carried out with the following primary antibodies: anti-P2X7R (Santa Cruz Biotechnology, Dallas, TX, 1:200), anti-mouse NLRP3 (Adipogen, San Diego, CA, 1:1000), anti-human NLRP3 (Cell Signaling Technology, Beverly, MA, 1:500), anti-ASC (Santa Cruz Biotechnology, Dallas, TX, 1:200), anti-IL-1β (Abcam, Cambridge, MA, 1:500), anti-mouse capsase-1 (Adipogen, 1:1000), anti-human caspase-1 (Cell Signaling Technology, 1:500), anti-human cleaved caspase-1 (Cell Signaling Technology, 1:500, only for Supplementary Fig. 5c), anti-ATP5a (Proteintech, Wuhan, China, 1:1000), anti-ATP5b (Proteintech, 1:1000), anti-Tubulin (Abcam, 1:2000), and anti-GAPDH (Cell Signaling Technology, 1:2000). After incubation with the appropriate horseradish peroxidase-conjugated IgG (Cell Signaling Technology), specific signals were determined on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai) by using an ECL kit (Thermo Scientific).
Qian Y., Qian C., Xie K., Fan Q., Yan Y., Lu R., Wang L., Zhang M., Wang Q., Mou S., Dai H., Ni Z., Pang H, & Gu L. (2021). P2X7 receptor signaling promotes inflammation in renal parenchymal cells suffering from ischemia-reperfusion injury. Cell Death & Disease, 12(1), 132.
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8

Quantification of Cellular PD-L1 Expression

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Cells were washed twice in phosphate-buffered saline (Beyotime, Shanghai, China), lysed in ice-cold radioimmune precipitation assay (Beyotime) buffer, and then centrifuged for 10 minutes at 4℃. Supernatant was collected, and protein concentrations were determined using the BCA Protein Assays (Invitrogen). Cell lysates were separated by SDS-PAGE (Beyotime) gel and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were blotted with 10% nonfat milk, washed in Trisbuffer saline (TBS) with 0.1% Tween (Beyotime) and incubated with primary polyclonal antibodies overnight at 4℃. After washing with TBS-Tween, membranes were incubated with secondary antibody (horseradish peroxidase conjugated IgG; Cell Signaling Technology, Danvers, USA) for 60 minutes at room temperature. Then, they were washed again with TBS-Tween and detected using the ChemiDoc Touch imaging system (Bio-Rad, Carlsbad, USA). PD-L1 (E1L3N®) XP® Rabbit mAb (1:1,000; Cell Signaling Technology) and β-Actin (13E5) Rabbit mAb #4970 (1:2,000; Cell Signaling Technology) were used for Western blotting.
Yang L., Cai Y., Zhang D., Sun J., Xu C., Zhao W., Jiang W, & Pan C. (2018). miR-195/miR-497 Regulate CD274 Expression of Immune Regulatory Ligands in Triple-Negative Breast Cancer. Journal of Breast Cancer, 21(4), 371-381.
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9

Western Blot Analysis of VEGF

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The protein was extracted in RIPA lysis buffer (200 μL, Thermo Scientific, USA). Equal amounts of protein from cell lysates were dissolved by 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore Corp., USA). After being blocked with 5% fat-free milk at room temperature for 2 h, the membranes were probed with primary antibody against VEGF (Genentech, Inc., CA) overnight at 4°C, and then incubated with secondary antibody horseradish peroxidase-conjugated IgG (Cell Signaling Technology, USA) at room temperature for 2 h. The protein was detected using an enhanced chemiluminescence kit (ECL kit, Pierce Biotechnology, IL) and the band intensity was quantified with software Image Pro Plus 4.5. GAPDH served as the loading control.
Shang L., Xu Y., Shao C., Ma C, & Feng Y. (2018). Anti-Vascular Endothelial Growth Factor (VEGF) Antibody Ameliorates Cartilage Degradation in a Rat Model of Chronic Sports Arthritic Injury. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 4073-4079.
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10

Immunoblotting for ResR/McdR Protein

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Immunoblotting was performed with anti-ResR/McdR antibodies that were commercially raised in rabbits using ResR/McdR-specific immunogenic peptides (ELASRRKHRDRKIG) (Genscript). Whole cell lysates were resolved by denaturing SDS-PAGE using 10% gel and transferred to nitrocellulose membrane by semi-dry electro-transfer method. The membrane was blocked with 5% non-fat dried milk (Bio-Rad) in 1× PBS containing 0.05% tween-20 (PBST) for 1 hour at 37 °C, followed by incubation with anti-ResR/McdR IgGs (0.2 µg/ml in the blocking buffer) for overnight at 4 °C. The membrane was washed thrice with 1× PBS containing 0.1% tween-20 followed by 1 hour of incubation with horseradish peroxidase-conjugated IgG (Cell Signaling Technology) in PBST. Extensive washing of the membrane was subsequently performed with PBST to remove unbound IgGs. Chemiluminescence signals were obtained after incubation of the blot with the Super Signal West Femto (Thermo Fisher) using a gel documentation system (Bio-Rad).
Pal P., Khan M.Y., Sharma S., Kumar Y., Mangla N., Kaushal P.S, & Agarwal N. (2023). ResR/McdR-regulated protein translation machinery contributes to drug resilience in Mycobacterium tuberculosis. Communications Biology, 6, 708.
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