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Mouse anti rabbit hrp

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Mouse anti-rabbit-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and visualize the presence of rabbit primary antibodies in various immunoassays and immunohistochemical applications.

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Lab products found in correlation

Human fibrinogen, by Enzyme Research (3 mentions) Prothrombin, by Enzyme Research (3 mentions) Glu plasminogen, by Enzyme Research (3 mentions) α thrombin, by Enzyme Research (3 mentions) Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions) Sds page gel, by Bio-Rad (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Amersham ecl plus western blotting detection system, by GE Healthcare (2 mentions) Goat anti rabbit hrp, by Jackson ImmunoResearch (2 mentions) Goat anti rat hrp, by Jackson ImmunoResearch (2 mentions) Goat anti mouse hrp, by Jackson ImmunoResearch (2 mentions) Caveolin 3, by BD (1 mentions) Flotillin 2, by BD (1 mentions) Ha tag, by Roche (1 mentions) Donkey anti guinea pig, by Jackson ImmunoResearch (1 mentions) Anti flag, by Thermo Fisher Scientific (1 mentions) Anti phospho pka substrate, by Cell Signaling Technology (1 mentions) Multipep rsi robot, by Intavis (1 mentions) Anti gst, by Santa Cruz Biotechnology (1 mentions) Recombinant human upa, by R&D Systems (1 mentions)

10 protocols using mouse anti rabbit hrp

1

Zika Virus Envelope Protein Expression

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To assess transgene expression from DNA vaccines, cell lysates obtained 48 h following lipofectamine 2000 (Invitrogen, CA, USA) transient transfection of 293T cells were mixed with reducing sample buffer, heated for 5 min at 100°C, cooled on ice, and run on a precast 4–15% SDS-PAGE gel (Biorad, CA, USA). Protein was transferred to PVDF membranes using the iBlot dry blotting system (Invitrogen, CA, USA), and the membranes were blocked overnight at 4°C in PBS-T (Dulbeco’s Phosphate Buffered Saline + 0.2% V/V Tween 20 + 5% W/V non-fat milk powder). Following overnight blocking, the membranes were incubated for 1 h with PBS-T containing a 1:5000 dilution of mouse anti-ZIKV Env mAb (BioFront Technologies, FL, USA). Membranes were then washed 3 times with PBS-T and incubated for 1 h with PBS-T containing a 1:1000 dilution of rabbit anti-mouse HRP (Jackson ImmunoResearch, PA, USA). Membranes were then washed 3 times with PBS-T and developed using the Amersham ECL plus Western blotting detection system (GE Healthcare, Chicago, USA).
Larocca R.A., Abbink P., Peron J.P., de A. Zanotto P.M., Iampietro M.J., Badamchi-Zadeh A., Boyd M., Ng’ang’a D., Kirilova M., Nityanandam R., Mercado N.B., Li Z., Moseley E.T., Bricault C.A., Borducchi E.N., Giglio P.B., Jetton D., Neubauer G., Nkolola J.P., Maxfield L.F., De La Barrera R.A., Jarman R.G., Eckels K.H., Michael N.L., Thomas S.J, & Barouch D.H. (2016). Vaccine Protection Against Zika Virus from Brazil. Nature, 536(7617), 474-478.
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2

Western Blot Analysis of Membrane Proteins

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Standard western blotting was carried out using 6%–20% gradient gels. The primary antibodies used were as follows: zDHHC5 (1:1,000, Sigma, HPA014670), NCX1 (1:1,000, Swant, R3F1), PLM (FXYD1, 1:1,000, Abcam, ab76597), Flotillin-2 (1:1,000, BD Biosciences, 610383), Caveolin-3 (1:4,000, BD Biosciences, 610420), HA-tag (1:5,000, Roche, 11867423001). The secondary antibodies used were as follows: Rabbit anti-mouse HRP (1:2,000, Jackson ImmunoResearch 111-035-144), Goat anti-rabbit HRP (1:2,000, Jackson ImmunoResearch 315-035-003), Goat anti-rat HRP (1:2,000, Jackson ImmunoResearch, 313-035-003), Donkey anti-guinea pig (1:2,000, Jackson ImmunoResearch, 106-035-003).
Main A., Boguslavskyi A., Howie J., Kuo C.W., Rankin A., Burton F.L., Smith G.L., Hajjar R., Baillie G.S., Campbell K.S., Shattock M.J, & Fuller W. (2022). Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies. Frontiers in Physiology, 13, 1023237.
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3

Peptide Array Synthesis and Protein Interactions

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Peptide arrays were synthesised in‐house on cellulose membrane supports via automatic SPOT synthesis using the MultiPep RSi Robot (Intavis, Tübingen, Germany) and 9‐fluorenylmethoxycarbonyl chloride (Fmoc) chemistry. Peptide sequences from DHX9 and PDE4D7 were spotted as 25‐mer peptides shifted by five amino acids, alongside alanine scans, N‐ and C‐terminal truncations and point substitutions to identify crucial amino acids for protein–protein interactions. For identification of PKA phosphorylation of DHX9, peptide arrays were incubated with or without 100 units of active bovine PKA catalytic subunit. For binding between DHX9 and PDE4D7, membranes were overlaid with either purified protein or overexpressing cell lysate. Far western blotting was then conducted with the following antibodies prior to chemiluminescent imaging: anti‐GST 1 : 1000 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc‐138), anti‐phospho‐PKA‐substrate 1 : 1000 (Cell Signalling, Danvers, MA, USA, #9624S), anti‐FLAG 1 : 1000 (ThermoFisher, #PA1‐984B), Goat‐anti‐rabbit HRP 1 : 2000 (Jackson ImmunoResearch, #111‐035‐144), Rabbit Anti Mouse HRP 1 : 2000 (Jackson ImmunoResearch, West Grove, PA, USA, 315‐035‐003).
Gulliver C., Busiau T., Byrne A., Findlay J.E., Hoffmann R, & Baillie G.S. (2024). cAMP‐phosphodiesterase 4D7 (PDE4D7) forms a cAMP signalosome complex with DHX9 and is implicated in prostate cancer progression. Molecular Oncology, 18(3), 707-725.
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4

Zika Virus Envelope Protein Expression

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To assess transgene expression from DNA vaccines, cell lysates obtained 48 h following lipofectamine 2000 (Invitrogen, CA, USA) transient transfection of 293T cells were mixed with reducing sample buffer, heated for 5 min at 100°C, cooled on ice, and run on a precast 4–15% SDS-PAGE gel (Biorad, CA, USA). Protein was transferred to PVDF membranes using the iBlot dry blotting system (Invitrogen, CA, USA), and the membranes were blocked overnight at 4°C in PBS-T (Dulbeco’s Phosphate Buffered Saline + 0.2% V/V Tween 20 + 5% W/V non-fat milk powder). Following overnight blocking, the membranes were incubated for 1 h with PBS-T containing a 1:5000 dilution of mouse anti-ZIKV Env mAb (BioFront Technologies, FL, USA). Membranes were then washed 3 times with PBS-T and incubated for 1 h with PBS-T containing a 1:1000 dilution of rabbit anti-mouse HRP (Jackson ImmunoResearch, PA, USA). Membranes were then washed 3 times with PBS-T and developed using the Amersham ECL plus Western blotting detection system (GE Healthcare, Chicago, USA).
Larocca R.A., Abbink P., Peron J.P., de A. Zanotto P.M., Iampietro M.J., Badamchi-Zadeh A., Boyd M., Ng’ang’a D., Kirilova M., Nityanandam R., Mercado N.B., Li Z., Moseley E.T., Bricault C.A., Borducchi E.N., Giglio P.B., Jetton D., Neubauer G., Nkolola J.P., Maxfield L.F., De La Barrera R.A., Jarman R.G., Eckels K.H., Michael N.L., Thomas S.J, & Barouch D.H. (2016). Vaccine Protection Against Zika Virus from Brazil. Nature, 536(7617), 474-478.
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5

Protease Activity Assay Protocol

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Human fibrinogen (plasminogen, von Willebrand factor, and fibronectin-depleted) and human zymogens Glu-plasminogen, α-thrombin, prothrombin, factor X zymogen, and activated factor Xa (all >95% purity) were purchased from Enzyme Research Laboratories, South Bend, IN, USA. Human plasmin and tranexamic acid (TXA) were purchased from MilliporeSigma, Burlington, MA, USA. Recombinant human pro-uPA zymogen [70 (link)] was kindly provided M. Ploug (Finsen Laboratory, Copenhagen, Denmark). Recombinant human uPA and recombinant mouse testisin proteins were from R&D Systems, Minneapolis, MN, USA. Fluorogenic substrates for thrombin (thrombin substrate III, Bz-FVR-AMC), FXa (Boc-IEGR-AMC [36 (link)]), uPA (Glt-GR-AMC [36 (link)]), plasmin (Boc-EEK-AMC [71 (link)]), and trypsin-like (Boc-QAR-AMC) proteases were from BACHEM, Bubendorf, Switzerland. Rivaroxaban (BAY 59-7939) was from Thermo Fisher Scientific, Waltham, MA, USA. Antibodies used were mouse anti-testisin antibody (MAbD9.1) [12 (link)], mouse anti-uPA antibody (IM15L, MilliporeSigma, Burlington, MA, USA), and rabbit anti-β-tubulin antibody (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies were goat anti-mouse-HRP, mouse anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA), and donkey anti-sheep-HRP (R&D Systems, Minneapolis, MN, USA).
Buzza M.S., Pawar N.R., Strong A.A, & Antalis T.M. (2023). Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin. International Journal of Molecular Sciences, 24(11), 9306.
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6

Antibody Characterization for Testisin and Cell Markers

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The anti-testisin monoclonal antibody MAbD9.1 was purified from the PTA-6077 hybridoma cell line (ATCC Pro104.D9.1) and characterized for both mouse and human anti-testisin specificity (S2 Fig), since available commercial anti-testisin antibodies were found not to specifically recognize murine or human testisin. Additional antibodies were rat anti-CD31 (BD 553370), rabbit anti-occludin (Invitrogen 71–1500), rabbit anti-ZO-1 (Invitrogen 61–7300), rabbit anti-claudin-5 (Invitrogen 34–1600), rabbit anti-NG-2 (Millipore AB5320), mouse anti-VE-cadherin (Santa Cruz sc-9989), rabbit anti-VE-cadherin (Abcam ab33168), rabbit anti-phospho (Tyr658)-VE-cadherin (Invitrogen 44-1144G), rabbit anti-β-catenin (Cell Signaling 8480), rabbit anti-GAPDH (Cell Signaling 2118), rabbit anti-β-actin (Cell Signaling 4970). Secondary antibodies were goat anti-mouse-HRP (Jackson ImmunoResearch 115-035-146), mouse anti-rabbit-HRP (Jackson ImmunoResearch 211-035-109), goat anti-rat-Alexa Fluor 488 (Invitrogen A11006), goat anti-mouse-Alexa Fluor 555 (Invitrogen A32727), goat anti-rabbit-Alexa Fluor 555 (Invitrogen A32732), goat anti-mouse-Alexa Fluor 488 (Invitrogen A32723).
Peroutka R.J., Buzza M.S., Mukhopadhyay S., Johnson T.A., Driesbaugh K.H, & Antalis T.M. (2020). Testisin/Prss21 deficiency causes increased vascular permeability and a hemorrhagic phenotype during luteal angiogenesis. PLoS ONE, 15(6), e0234407.
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7

Western Blot Protein Detection

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Following cell lysis, protein concentration was assessed using the Bio-Rad DC Protein Assay (Bio-Rad:500–0111) and protein (2–40 μg) was resolved by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PDVF) membranes (Immunobilon Transfer Membranes:IPVH00010). Transferred membranes were blocked with 5% milk for 30 minutes and probed with appropriate antibody in 1% milk solution for either 1 hour at room temperature or 12 hours at 4°C, with three 10 min washes with 1XPBS-0.05% Tween 20 between each antibody incubation. Antibodies included: Anti-Stau1 [1:1000] (Abcam:ab73478), Anti-GAPDH [1:10,000] (Abcam:ab8245), Anti-β-Actin [1:500] (Santa Cruz:sc-47778), Anti-CUGBP1 [1:1000] (Santa Cruz:sc-20003), Anti-hnRNP H [1:5000] (Abcam:10374), Anti-MyoD [1:300] (BD Pharmingen: 554130), Anti-MBNL1 antibody [1:300] (Abnova:H00004154), Anti-HA F7 probe [1:1000] (Santa Cruz:sc-7392). Secondary antibodies included: Mouse-anti-Rabbit HRP [1:20,000] (Jackson ImmunoResearch:211-032-171) and Goat-anti-Rabbit HRP [1:10,000] (Molecular Probes:MP 02764). Proteins on membranes were detected with Millipore-Luminata Crescendo Western HRP Substrate (WBLUR0500) and visualized on film (HyBlot CL Autoradiography Film:E3018).
Bondy-Chorney E., Crawford Parks T.E., Ravel-Chapuis A., Klinck R., Rocheleau L., Pelchat M., Chabot B., Jasmin B.J, & Côté J. (2016). Staufen1 Regulates Multiple Alternative Splicing Events either Positively or Negatively in DM1 Indicating Its Role as a Disease Modifier. PLoS Genetics, 12(1), e1005827.
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8

Western Blot Antibody Quantification

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Western blot antibodies were used at the following dilutions: rabbit-anti-Me31B (1:20,000), mouse-anti-Cup (1:2,000), rabbit-anti-Tral (1:2,000), rat-anti-Tral (1:4,000), rabbit-anti-GFP (1:100,000), rat-anti-Vas (1:2,000), and mouse-anti-α-Tubulin (1:100,000). Secondary antibodies were used at the following dilutions: mouse-anti-rabbit HRP (Jackson ImmunoResearch) (1:10,000 for rabbit-anti-Me31B primary antibody, 1:5,000 for rabbit-anti-Tral, 1:10,000 for rabbit-anti-GFP), goat-anti-mouse HRP (Santa Cruz Biotechnology) (1: 50,000 for mouse-anti-α-Tubulin primary antibody, 1:5,000 for mouse-anti-Cup primary antibody), goat-anti-rat HRP (Jackson ImmunoResearch) (1:10,000 for rat-anti-Tral primary antibody, and 1:5,000 for rat-anti-Vas). The protein band quantification analysis was performed by using ImageJ (https://imagej.nih.gov/ij/).
McCambridge A., Solanki D., Olchawa N., Govani N., Trinidad J.C, & Gao M. (2020). Comparative Proteomics Reveal Me31B’s Interactome Dynamics, Expression Regulation, and Assembly Mechanism into Germ Granules during Drosophila Germline Development. Scientific Reports, 10, 564.
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9

Molecular Mechanisms of Ketamine Action

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Ketamine hydrochloride injection was purchased from Fujian Gutian Pharmaceutical Co., Ltd. (China). Bovine brain-heart infusion broth was bought from OXIOD (UK). BASO rapid Gram stain was obtained from Zhuhai Beisuo Biological Technology Co., Ltd. (China). Rb antinuclear factor-κB (NF-κB) p65, Rb antiphospho-c-Jun N-terminal kinase (JNK), and Rb antiphospho-p38 mitogen-activated protein kinase (MAPK) were purchased from Bio Basic Inc. Mouse-anti-rabbit HRP was bought from Jackson. ELISA kit was obtained from Uscn Life Science, Inc. (China). Real-time PCR reagent was purchased from Invitrogen Life Technologies (USA).
Lin G., Chen S., Lei L., You X., Huang M., Luo L., Li Y., Zhao X, & Yan F. (2015). Effects of Intravenous Injection of Porphyromonas gingivalis on Rabbit Inflammatory Immune Response and Atherosclerosis. Mediators of Inflammation, 2015, 364391.
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10

Western Blot Analysis of APP in APP/PS1 Mice

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Hippocampus from APP/PS1 mice treated with Pimavanserin for 12 hr was lysed in 1% Triton X-100, 0.1% SDS in PBS by sonication. Twenty microgram of total protein was loaded per lane on SDS-PAGE gels. SDS-PAGE was performed using 16% Novex Tricine gels (Cat# EC66955, Invitrogen) under reducing conditions. Samples were loaded onto the gels in alternating basal and drug-treated samples. Nitrocellulose blots were blocked in 3% bovine serum albumin and probed with either rabbit-anti-APP antibody directed against the C-terminus (Cat# SAB3500274, Sigma-Aldrich) or mouse-anti-GAPDH (Cat# G8795, Sigma-Aldrich) followed by a mouse-anti-rabbit HRP (Cat# 211-032-171, Jackson ImmunoResearch) or a sheep-anti-mouse antibody (Cat# 45-000-692, GE Life Sciences) in 1% bovine serum albumin. Blots were washed in 0.05% Tween-20 in PBS three times for at least 5 min each. Bands were detected with Lumigen-TMA6 (Cat# TMA, Amersham) and captured digitally using the Kodak ImageStation 440CF. Densitometry was performed using the Kodak 1D Image Analysis software. Tissue protein levels were normalized to GAPDH for each lane, then normalized to % vehicle for each blot, followed by data from two blots being combined for analysis.
Yuede C.M., Wallace C.E., Davis T.A., Gardiner W.D., Hettinger J.C., Edwards H.M., Hendrix R.D., Doherty B.M., Yuede K.M., Burstein E.S, & Cirrito J.R. (2021). Pimavanserin, a 5HT2A receptor inverse agonist, rapidly suppresses Aβ production and related pathology in a mouse model of Alzheimer’s disease. Journal of neurochemistry, 156(5), 658-673.
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