Hrp conjugated anti mouse igg

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

HRP-conjugated anti-mouse IgG is a lab equipment product that is used as a secondary antibody in various immunoassay techniques. It consists of horseradish peroxidase (HRP) enzyme conjugated to an antibody that specifically binds to mouse immunoglobulin G (IgG) molecules.

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Close spelling variants of this product

HRP anti-mouse (4 citations) IgG (Mouse) (3 citations) Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit and anti-mouse IgGs (2 citations)

108 protocols using hrp conjugated anti mouse igg

Antibody-based NADPH Oxidase Assay

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Antibodies used include the following: anti-APE1, mouse monoclonal anti-Nox1 (Novus Biologicals), rabbit polyclonal anti-APE1, mouse monoclonal anti-Rac1 clone 28A (Millipore) followed by incubation with anti-rabbit or anti-mouse HRP-conjugated IgG (Cell Signaling Technology). NADPH oxidase inhibitor diphenyleneiodonium (DPI) and Rac inhibitor NSC23766 were purchased from Calbiochem.

den Hartog G., Chattopadhyay R., Ablack A., Hall E.H., Butcher L.D., Bhattacharyya A., Eckmann L., Harris P.R., Das S., Ernst P.B, & Crowe S.E. (2016). Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia. PLoS Pathogens, 12(1), e1005382.

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Cell Cycle Regulation by DAP3 Knockdown

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Calcium- and magnesium-free phosphate-buffered saline PBS(−) and paclitaxel (#169-18 611) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Propidium iodide (PI) and the chk2 inhibitor II were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). SCH900776 was purchased from Med Chem Express (Shanghai, China). The anti-rabbit horseradish peroxidase (HRP)-conjugated IgG (#7074), and the anti-mouse HRP-conjugated IgG (#7076) secondary antibodies, as well as the anti-phospho-cdc2 (Tyr15) (#9111), the anti-cyclin B1 (#4135), the anti-phospho-histone H3 (Ser10) XP (#3377), the anti-phospho-chk1 (Ser296) (#2349), the anti-chk1 (#2360), the anti-phospho-chk2 (Thr68) (#2661), the anti-p21 (#2947) and the anti-β-actin (#4967) monoclonal antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). The anti-DAP3 (Cat. No. 610662) monoclonal primary antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The Ambion Silencer® Select Pre-designed siRNA against the gene-encoding DAP3 (Cat. No. s1506) and the Silencer® Select Negative #1 Control (Cat. No. AM4611) siRNAs were purchased from Thermo Fisher Scientific, Inc (Waltham, MA, USA).

Sato Y., Yoshino H., Sato K., Kashiwakura I, & Tsuruga E. (2023). DAP3-mediated cell cycle regulation and its association with radioresistance in human lung adenocarcinoma cell lines. Journal of Radiation Research, 64(3), 520-529.

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Protein Extraction and Western Blot Analysis

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Protein was extracted from cells or tissues using T-per tissue protein extraction reagent (catalog #78510) lysis buffer. Concentration was measured using the Bradford Assay reagent. 10-40 ug of protein was loaded on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blotted with anti-Mettl3 (#ab195352 Abcam, #MA5-27527 Invitrogen), anti-Mettl14 (#HPA038002, Sigma), anti-GFP (#GFP-1020 Aves Lab), anti-HA (#3724, Cell Signaling), anti-c-Myc (#ab185656 Abcam, #OP10-200ug Sigma), anti-Avpr2 (#AB1797P, EMD Millipore), anti-Yap1 (#4912, Cell Signaling), anti-pCREB (#9198S, pCreb1), anti-PCNA (#sc-7907, Santa Cruz), or anti-puromycin (#PMY-2A4, Developmental Studies Hybridoma Bank) antibodies. All the primary antibodies were used at 1:1000 dilution, except for anti-puromycin, which was used at 1:50 dilution. Goat anti-rabbit-HRP conjugate (#7074S, Cell Signaling) or anti-mouse HRP-conjugated IgG (#7076S, Cell Signaling) was used as the secondary antibody. HRP-conjugated Actin antibody (#A3865, Sigma) was used at 1:40,000 dilution for measuring total protein. The blots were developed using the X-ray film developer or the Bio-rad digital imager. The protein bands were quantified using the Imagelab software from Bio-rad. Each western blot was repeated at least three times.

Ramalingam H., Kashyap S., Cobo-Stark P., Flaten A., Chang C.M., Hajarnis S., Hein K.Z., Lika J., Warner G.M., Espindola-Netto J.M., Kumar A., Kanchwala M., Xing C., Chini E.N, & Patel V. (2021). A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease. Cell metabolism, 33(6), 1234-1247.e7.

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Western Blot Analysis of TLR Proteins

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NK whole-cell extracts were quantified by a bicinchoninic acid assay (BCA) (Thermo Fisher Scientific), resolved on 8% SDS-PAGE, and transferred onto nitrocellulose membrane. Filters were probed with primary antibodies overnight at 4°C, followed by biotinylated anti-mouse IgG (H+) (1:1000) or biotinylated anti-rabbit IgG (H+) (1:7500) (Vector Laboratories, Burlingame, California, USA) for 30 min at room temperature. Finally, filters were incubated for 30 min at room temperature with streptavidin-horseradish peroxidase (HRP) conjugate diluted 1:1000 in Tris-NaCl-Tyramide Signal Amplification (TNB-TSA) blocking buffer (0.1 M Tris–HCl, pH 7.5; 0.15M NaCl; 0.5% TSA blocking reagent) (PerkinElmer, Waltham, Massachusetts, USA). The following primary monoclonal antibodies were used: TLR3 (TLR3.7) (Thermo Fisher Scientific); TLR7 (4G6) (Bio-Techne, Abingdon, UK); TLR8 (D3Z6J) and TLR9 (D9M9H) XP rabbit monoclonal antibodies (Cell Signaling Technologies). β-actin (C4, Santa Cruz Biotechnology, Dallas, Texas, USA) was used as housekeeping. To detect β-actin, filter was further incubated with anti-mouse HRP-conjugated IgG (Cell Signaling Technologies).

Veneziani I., Alicata C., Pelosi A., Landolina N., Ricci B., D'Oria V., Fagotti A., Scambia G., Moretta L, & Maggi E. (2022). Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16− subset. Journal for Immunotherapy of Cancer, 10(1), e003385.

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Extracellular Vesicle Protein Analysis

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To extract proteins, colosEVs were lysed in RIPA Buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Total protein concentration was measured using Bradford assay. A quantity of 25 µg of total proteins were separated using 12% T sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Blotted membranes were saturated and incubated overnight with primary antibodies against CD81 (1:500; Bioss Antibodies, Woburn, MA, USA), TSG-101 (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and calnexin (1:400 sc-23954, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-rabbit HRP-conjugated IgG (1:3000, Cell Signaling Technology, Danvers, MA, USA) and anti-mouse HRP-conjugated IgG (1:3000, Cell Signaling Technology) were used as secondary antibody. Clarity Western ECL Substrate (Bio-Rad) was used to evidence protein bands, and the images were acquired using a GS-800 imaging systems scanner (Bio-Rad, Hercules, CA, USA).

Mecocci S., De Paolis L., Zoccola R., Fruscione F., De Ciucis C.G., Chiaradia E., Moccia V., Tognoloni A., Pascucci L., Zoppi S., Zappulli V., Chillemi G., Goria M., Cappelli K, & Razzuoli E. (2022). Antimicrobial and Immunomodulatory Potential of Cow Colostrum Extracellular Vesicles (ColosEVs) in an Intestinal In Vitro Model. Biomedicines, 10(12), 3264.

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Cloning and Detection of FMNL2 and FMNL3

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Human cDNAs coding for FMNL2 (Q96PY5-3) and FMNL3 (Q8IVF7-3) were purchased from Promega (Madison, WI, USA). ECL Prime Western Blotting Detection Reagent was sourced from GE Healthcare (Amersham, UK). The dye terminator cycle sequencing kit, Lipofectamine LTX with PLUS reagent and Hoechst 33342 were obtained from Life Technologies Corp. (Carlsbad, CA, USA). Anti-FLAG monoclonal antibody and anti-mouse IgG-FITC antibody were obtained from Sigma (St. Louis, MO, USA). Tetradec-13-ynoic acid (Alk-Myr) was sourced from Cayman Chemical Co. (Ann Arbor, MI, USA). 5-TAMRA Azide (Az-TAMRA) was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) were obtained from Sigma (St. Louis, MO, USA). Protein G-HRP conjugate was sourced from Bio-Rad (Hercules, CA, USA). HRP-conjugated anti-mouse IgG was purchased from Cell Signaling Technology (Danvers, MA, USA). The other reagents used were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) or Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan), and were of analytical or DNA grade.

Kinoshita-Kikuta E., Tanikawa A., Hosokawa T., Kiwado A., Moriya K., Kinoshita E., Koike T, & Utsumi T. (2019). A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE. PLoS ONE, 14(11), e0225510.

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Quantification of Methylglyoxal-Protein Adducts

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Total protein content of the extracts was determined using the Bradford protein assay (Bio-Rad, Segrate (MI), Italy) with bovine serum albumin (Sigma Aldrich, Milano, Italy) as standards. Fifty micrograms of each protein extract were diluted with Laemmli buffer and incubated at 100 °C for 5 min. Subsequently, all samples were loaded onto a 12% SDS-polyacrylamide gel and electrophoresed at 80 V. After electrophoretic separation, proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo (Bio-Rad, Segrate (MI), Italy) protein transfer system. Membranes were first blocked in 5% nonfat milk / 1X TBS-T and then incubated over night at 4 °C, with a 1:500 dilution in the same blocking buffer, of mouse anti- methylglyoxal-protein adducts. After washes, membranes were incubated with 1:2000 dilution of HRP-conjugated anti-mouse IgG (Cell Signaling Technology, Pero (MI), Italy). Immunocomplexes were detected by using the SuperSignal West Femto ECL substrate (Pierce, Pero (MI), Italy). Fully automated densitometric software, Alliance 2.7 1D (UVITEC, Eppendorf, Milan, Italy), was used for acquisition and analysis of immunoblots images.

Coluccio M.L., Gentile F., Presta I., Donato G., Coppedè N., Valprapuram I., Mignogna C., Lavecchia A., Figuccia F., Garo V.M., Fabrizio E.D., Candeloro P., Viglietto G, & Malara N. (2020). Tailoring Chemometric Models on Blood-Derived Cultures Secretome to Assess Personalized Cancer Risk Score. Cancers, 12(6), 1362.

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Western Blotting of Muscle Differentiation Markers

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Cells were lysed in modified RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% IGEPAL, protease and phosphatase inhibitors) and 10–30μg of total protein was loaded into 10% SDS-PAGE gels and transferred to PVDF membranes for Western blotting with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7, 1:10; mouse monoclonal (F5D) anti-myogenin (Developmental Studies Hybridoma Bank, USA), 1:5; mouse monoclonal anti-tubulin, 1:10000; mouse monoclonal anti-HA HRP conjugated, 1:4000 (Sigma-Aldrich,USA); mouse monoclonal anti-HDAC2 [3F3], 1:5000; rabbit polyclonal ChiP-grade anti-HA tag, 1:10000; rabbit polyclonal anti-Nedd4, 1:10000; rabbit monoclonal anti-GFP E385, 1:5000 (Abcam, UK); mouse monoclonal anti-GAPDH (EMD-Millipore, USA), 1:10000; mouse monoclonal 9B11 myc-tag (Cell Signaling, USA), 1:1000; rabbit polyclonal anti-GST (gift from Dr. María Paz Marzolo, Santiago, Chile), 1:5000 and mouse monoclonal P4D1 anti-ubiquitin (Santa Cruz Biotechnology, USA), 1:500. As secondary antibodies HRP conjugated anti-mouse IgG and anti-rabbit IgG (Cell Signaling, USA) were used at 1:5000. HRP activity was detected using the SuperSignal West Dura Extended Duration Substrate (Thermo-Fisher Scientific, USA). When indicated, lysates/fractions were subjected to co-immunoprecipitation as described [16 (link)].

Bustos F., de la Vega E., Cabezas F., Thompson J., Cornelison D., Olwin B.B., Yates JR I.I.I, & Olguín H.C. (2015). NEDD4 REGULATES PAX7 LEVELS PROMOTING ACTIVATION OF THE DIFFERENTIATION PROGRAM IN SKELETAL MUSCLE PRECURSORS. Stem cells (Dayton, Ohio), 33(10), 3138-3151.

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Western Blot Analysis of Protein Expression

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Whole cell lysates were collected using RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 0.5 mM EGTA; 1% Igepal CA-630; 0.5% sodium deoxycholate; and 0.1% SDS) and protein concentration was determined through Bio-Rad DC protein assay (Bio-Rad). 10 μg of proteins were resolved on precast 4–12% SDS-polyacrylamide gels (M00654, GeneScript) and transferred onto PVDF membranes (#88518, Thermo Fisher Scientific). The membranes were then stained with Ponceau Red, blocked for 1h in TBS-T buffer containing 1% Tween-20 with 5% non-fat dry milk, and incubated with primary antibodies overnight at 4°C. The primary antibodies were directed against Iba-1 (1:1000) (ab178847, Abcam, Cambridge, MA), DDX3 (1:3000) (sc-365768, Santa Cruz Biotechnology, Dallas, Texas) and GAPDH (1:10,000) (Cell Signaling Technology, Danvers, MA). Secondary antibodies were HRP-conjugated anti-mouse IgG (1:8000; 7076S), or HRP-conjugated anti-rabbit IgG (1:8000; 7074S), both from Cell Signaling. Membranes were processed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).

Aksenova M., Sybrandt J., Cui B., Sikirzhytski V., Ji H., Odhiambo D., Lucius M.D., Turner J.R., Broude E., Peña E., Lizarraga S., Zhu J., Safro I., Wyatt M.D, & Shtutman M. (2019). Inhibition of the Dead Box RNA Helicase 3 prevents HIV-1 Tat and cocaine-induced neurotoxicity by targeting microglia activation. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 15(2), 209-223.

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Immunoblotting and Immunofluorescence Analysis of Extracellular Vesicle Proteins

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The antibodies used for immunoblotting and immunofluorescence were as follows: Cortactin (H222) (3503; Cell Signaling Technology), VAMP7 (NB100–91356; Novus Biologicals), RAB7 (D95F2) (9367 T; Cell Signaling Technology), RAB27B (13412–1-AP; Proteintech), CD63 (GTX28219; GeneTex), β-actin (20536–1-AP; Proteintech), TSG101(28283–1-AP; Proteintech), CD81(A5270; ABclonal), SNAP23 (ab4114; Abcam), HRP-conjugated anti-rabbit IgG (7074S; Cell Signaling Technology), and HRP-conjugated anti-mouse IgG (7076S; Cell Signaling Technology). The reagent used for immunoblotting was Phalloidin-iFluor 594 reagent (ab176757; Abcam).

Peng X., Li X., Yang S., Huang M., Wei S., Ma Y., Li Y., Wu B., Jin H., Li B., Tang S., Fan Q., Liu J., Yang L, & Li H. (2021). LINC00511 drives invasive behavior in hepatocellular carcinoma by regulating exosome secretion and invadopodia formation. Journal of Experimental & Clinical Cancer Research : CR, 40, 183.

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