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Donkey anti rabbit hrp

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Donkey anti-rabbit-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and amplify the signal from rabbit primary antibodies in various immunoassays and immunohistochemical applications.

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Lab products found in correlation

Sheep anti mouse hrp, by GE Healthcare (8 mentions) Mouse anti gfp, by Roche (2 mentions) Goat anti mouse hrp, by GE Healthcare (2 mentions) Imagequant las 4000, by GE Healthcare (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bca protein assay, by Thermo Fisher Scientific (2 mentions) Mouse anti flag m2, by Merck Group (1 mentions) Irdye 800cw donkey anti mouse, by LI COR (1 mentions) Irdye 680rd donkey anti rabbit, by LI COR (1 mentions) Odyssey clx infrared imaging system, by LI COR (1 mentions) Image studio 5, by LI COR (1 mentions) Mouse anti ha ha 11, by Fortrea (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Oligomycin, by Merck Group (1 mentions) 7 azido 4 methylcoumarin, by Merck Group (1 mentions) Adenosine 5 triphosphate atp disodium salt hydrate, by Merck Group (1 mentions) N n diethyl p phenylenediamine sulfate, by Merck Group (1 mentions) Iron 3 chloride fecl3, by Merck Group (1 mentions) Potassium phosphate monobasic, by Merck Group (1 mentions) Magnesium chloride, by Merck Group (1 mentions)

21 protocols using donkey anti rabbit hrp

1

Quantitative Western Blot Protocol

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For western immunoblotting, samples were fixed in TCA, acetone-washed and whole cell extracts prepared by bead-beating in TE containing protease inhibitors before running on SDS-PAGE and transferring to nitrocellulose membrane. Antibodies used were mouse anti-Ha (HA11, Covance) at 1:1000 dilution, mouse anti-GFP (Roche) at 1:1000, mouse anti-V5 (SV5-Pk1, Bio-Rad) at 1:2000, mouse anti-Flag (M2, Sigma) at 1:1000, rabbit anti-Pgk1 (lab stock) at 1:10000, rabbit anti-Kar2 (lab stock) at 1:20000, sheep anti-mouse-HRP (GE Healthcare) at 1:5000, donkey anti-rabbit-HRP (GE Healthcare) at 1:10000, donkey anti-mouse-IRDye 800CW (LI-COR Biosciences) at 1:10000 and donkey anti-rabbit-IRDye 680RD (LI-COR Biosciences) at 1:10000. Quantitative western blotting was performed using an Odyssey CLx Infrared Imaging System (LI-COR Biosciences) and quantified using ImageStudio 5.2.5 (LI-COR Biosciences).
Galander S., Barton R.E., Borek W.E., Spanos C., Kelly D.A., Robertson D., Rappsilber J, & Marston A.L. (2019). Reductional Meiosis I Chromosome Segregation Is Established by Coordination of Key Meiotic Kinases. Developmental Cell, 49(4), 526-541.e5.
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2

Mitochondrial Hydrogen Sulfide Donor Synthesis

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AP39, a novel mitochondria-targeted H2S donor, was designed and synthesized by Medicilon Inc., as described previously [19 (link), 20 (link)]. Antimycin A, 0.25% trypsin, 2-deoxyglucose, oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), trichloroacetic acid, rotenone, 7-azido-4-methylcoumarin (AzMC), monobasic potassium phosphate, dibasic sodium succinate hexahydrate, adenosine 5′-triphosphate (ATP) disodium salt hydrate, fatty acid-free BSA, sodium hydrosulfide hydrate (NaSH·xH2O), N,N-diethyl-p-phenylenediamine sulfate, magnesium chloride, and iron(III) chloride (FeCl3) were purchased from Sigma-Aldrich Company. The monoclonal anti-Aβ antibody 6E10 was obtained from Covance Inc. (Princeton, NJ, USA). Drp1 was purchased from Novus Biologicals, Inc. Fis1 was purchased from Proteintech Group. Mfn1 and Mfn2 antibodies were purchased from Santa Cruz Biotech, and OPA-1 antibody was purchased from BD Transduction Laboratories. The secondary antibodies goat anti-mouse-HRP and donkey anti-rabbit-HRP antibodies were purchased from GE Healthcare. The Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM), 1% glutamine, and 1% penicillin were obtained from Invitrogen. All other chemicals were purchased from Amresco (Solon, OH, USA).
Zhao F.L., Fang F., Qiao P.F., Yan N., Gao D, & Yan Y. (2016). AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons. Oxidative Medicine and Cellular Longevity, 2016, 8360738.
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3

Antibody Validation for Western Blotting

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The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit Anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).
Kruse T., Gnosa S.P., Nasa I., Garvanska D.H., Hein J.B., Nguyen H., Samsøe‐Petersen J., Lopez‐Mendez B., Hertz E.P., Schwarz J., Pena H.S., Nikodemus D., Kveiborg M., Kettenbach A.N, & Nilsson J. (2020). Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits. The EMBO Journal, 39(13), e103695.
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4

Western Blot Analysis of Protein Signaling

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Proteins were extracted from glomeruli or podocytes with lysis buffer and were quantified by BCA protein assay (iNtRON Biotechnology). Samples were resolved on 4–12% Bis-Tris Criterion XT gels (Bio-Rad) then transferred to a polyvinylidene difloride membrane. Membranes were incubated with the appropriate primary antibodies: rabbit anti-phospho-EGFR (Tyr1068) (D7A5, Cell Signaling Technology, 1:1000), rabbit anti-EGFR (D38B1, Cell Signaling Technology, 1:1000), rabbit anti-STAT3 (D1A5, Cell Signaling Technology, 1:1000), rabbit anti-phospho-STAT3 (Tyr705) (D3A7, Cell Signaling Technology, 1:1000), goat anti-p57Kip2 (Clone M-20, Santa Cruz Biotechnology, 1:500). Protein loading was monitored by the rat anti-tubulin antibody (ab6160, Abcam, 1:5000). Secondary antibodies were donkey anti-rabbit HRP and donkey anti-goat HRP (GE Healthcare Life Sciences) with no cross reaction to sheep serum. Antigens were detected by enhanced chemiluminescence (Supersignal West Pico, Pierce) using a LAS-4000 imaging system (Fuji). Densitometric analysis with ImageJ software was used for quantification. The uncropped versions of western blotting are shown in Supplementary Fig. 10.
Henique C., Bollée G., Loyer X., Grahammer F., Dhaun N., Camus M., Vernerey J., Guyonnet L., Gaillard F., Lazareth H., Meyer C., Bensaada I., Legrès L., Satoh T., Akira S., Bruneval P., Dimmeler S., Tedgui A., Karras A., Thervet E., Nochy D., Huber T.B., Mesnard L., Lenoir O, & Tharaux P.L. (2017). Genetic and pharmacological inhibition of microRNA-92a maintains podocyte cell cycle quiescence and limits crescentic glomerulonephritis. Nature Communications, 8, 1829.
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5

Comprehensive Antibody Validation Protocol

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beta‐actin‐HRP (AC‐15, Sigma‐Aldrich), FLAG‐HRP (clone M2, Sigma‐Aldrich), V5‐HRP (R961‐25, Invitrogen), RIG‐I (Alme1, Calteg Medsystems), VZV‐gE/GI (MAB8612, GE Healthcare), VZV ORF62 (C05107MA, Meridian Life Science), pSTAT1 (Y701) (58D6, CellSignaling Technology), STAT1 (42H3, CellSignaling Technology), pSTAT2 (D3P2P, CellSignaling Technology), STAT2 (D9J7L, CellSignaling Technology), hcGAS (D1D3G, CellSignaling Technology), hSTING (D2P2F, CellSignaling Technology), MyD88 (D80F5, CellSignaling Technology), TBK1 (D1B4, CellSignaling Technology), MAVS (ALX‐210‐929‐C100, ENZO Life Science), IRF3 (D6I4C, CellSignaling Technology), VZV ORF9 (polyclonal rabbit serum, kind gift from Catherine Sadzot (University of Liege, Belgium) (Riva et al, 2013 (link)), donkey‐anti‐mouse‐HRP (NA931, GE Healthcare), donkey‐anti‐rabbit‐HRP (NA934, GE Healthcare).
Hertzog J., Zhou W., Fowler G., Rigby R.E., Bridgeman A., Blest H.T., Cursi C., Chauveau L., Davenne T., Warner B.E., Kinchington P.R., Kranzusch P.J, & Rehwinkel J. (2022). Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS. The EMBO Journal, 41(14), e109217.
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6

Antibody Validation for Western Blot and IF

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Antibodies for Western blot and immunofluorescence staining were purchased as follows: PAK1, PAK2, PAK3, PAK4, phospho-PAK4/5/6 (S474, S602, S560), PARP1, ATM, DNA-PK, PAK5, phospho-DNA PKs (S2056), and PAK6 (all Cell Signaling Technology, Frankfurt am Main, Germany); β-actin (Sigma-Aldrich); phospho-ATM (S1981) (Rockland Immunochemicals, Gilbertsville, PA, USA); Rad51 and γH2AX (both Merck Millipore, Burlington, MA, USA), 53BP1 (Novus Biologicals, Wiesbaden Nordenstadt, Germany); goat anti-mouse AlexaFluor488 and goat anti-rabbit AlexaFluor546 (both Life Technologies, Carlsbad, CA, USA); donkey anti-rabbit HRP and sheep anti-mouse HRP (both GE Healthcare, Chicago, IL, USA).
Blankenstein L.J., Cordes N., Kunz-Schughart L.A, & Vehlow A. (2022). Targeting of p21-Activated Kinase 4 Radiosensitizes Glioblastoma Cells via Impaired DNA Repair. Cells, 11(14), 2133.
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7

Immunoblotting Secondary Antibodies

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Donkey anti-Rabbit-HRP (GE Life Sciences #NA934, 1:5,000), Goat anti-mouse-HRP (GE Life Sciences #NA931, 1:5000), Goat anti-Rabbit IR Dye 800CW (LI-COR # 926–32211).
Courtland J.L., Bradshaw T.W., Waitt G., Soderblom E.J., Ho T., Rajab A., Vancini R., Kim I.H, & Soderling S.H. (2021). Genetic disruption of WASHC4 drives endo-lysosomal dysfunction and cognitive-movement impairments in mice and humans. eLife, 10, e61590.
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8

Immunoblotting of Nuclear Proteins

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The following abs were used at the indicated final concentration or dilution: mouse monoclonal abs to hnRNP U (Santa Cruz; 3G6; 0.2 μg/ml), hnRNP A1 (Sigma; 4B10; 2 μg/ml), lamin B1 (Calbiochem; 101-B7; 1/1,000), α-tubulin (Sigma; B-5-1-2; 1/1,000), G3BP1 (BD; 23/G3BP; 0.25 μg/ml), hnRNP C1/C2 (Sigma; 4F4; 0.4 μg/ml), β-actin (Developmental Studies Hybridoma Bank; 1/1,000), HuR (Santa Cruz; 3A2; 0.2 μg/ml); rabbit antisera to HMGB2 (Abcam; 1/1,000), ICAD (Abcam; 0.5 μg/ml), NE (Abcam; 1 μg/ml); DDX5 (Abcam; 0.5 μg/ml). Secondary abs were sheep anti-mouse-HRP (GE; 1/2,500), donkey anti-rabbit-HRP (GE; 1/2,500), donkey anti-goat-HRP (GE, 1/2,500), goat anti-rabbit AF488 (Invitrogen; 1/200), donkey anti-mouse Cy3 (Jackson ImmunoResearch 715-165-150; 1:200). Normal rabbit IgG (Cell Signaling 2729; 1 μg/ml) was used as an isotype control for immunofluorescence.
Thomas M.P., Whangbo J., McCrossan G., Deutsch A., Martinod K., Walch M, & Lieberman J. (2014). Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5390-5397.
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9

MCMV Immediate-Early Protein Analysis

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3T3 cells were infected with MCMV-m166HA. To selectively analyze immediate-early protein expression, cycloheximide (100 µg/ml; Sigma) was added to 3T3 cells 30 min prior to infection and was let sit on cells for up to 3 h post infection. At this time, cycloheximide media was washed away and actinomycin D (2.5 µg/ml; Sigma) was added to cells for another 3 h. To block late protein expression, Foscarnet (250 µg/ml; Sigma) was added just prior to infection and was present throughout. At 48 h after infection, cell pellets were scarped and lysed in protein sample buffer (3% SDS, 2% β-mercaptoethanol, 200 mM Tris [pH 8.8], 0.5M sucrose, 5 mM EDTA), boiled for 5 min, separated by SDS-PAGE (10% gel) and transferred to nitrocellulose filters. Filters were probed with anti-HA (H6908; Sigma) at 2 µg/ml followed by detection with donkey anti-rabbit HRP (GE healthcare). Signals were detected with the ECL detection kit (Amersham).
Verma S., Loewendorf A., Wang Q., McDonald B., Redwood A, & Benedict C.A. (2014). Inhibition of the TRAIL Death Receptor by CMV Reveals Its Importance in NK Cell-Mediated Antiviral Defense. PLoS Pathogens, 10(8), e1004268.
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10

Quantification of ERK and pERK Levels

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For quantification of ERK and pERK levels, 50 ventral ganglia from late third instar larvae were dissected in ice-cold fixation solution (4% paraformaldehyde in PBS) to minimize changes in phosphorylation status during handling, fixed for 10 min on ice and washed three times for 20 min in PBS. Following homogenization and sonication, protein lysates were analyzed by western blotting with the following antibodies: monoclonal rabbit anti-Phospho-p44/42 (ERK1/2, Thr202/Tyr204; clone D13.14.4E, 1:2000; Cell Signaling), monoclonal rabbit anti-p44/42 (ERK1/2; clone 37F5, 1:1000; Cell Signaling) and mouse anti-α-tubulin (clone DM1a, 1:10,000; Sigma-Aldrich). Sheep anti-mouse-HRP (1:10,000; GE Healthcare) and donkey anti-rabbit-HRP (1:10,000; GE Healthcare) were used as secondary antibodies. Western blots were scanned with an Intas ChemoCam imager, and intensities of protein bands were measured with the ImageJ 1.48a software (NIH, Bethesda, MD, USA). The intensity of the α-tubulin band was used for normalization.
Beck K., Ehmann N., Andlauer T.F., Ljaschenko D., Strecker K., Fischer M., Kittel R.J, & Raabe T. (2015). Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons. Disease Models & Mechanisms, 8(11), 1389-1400.
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