Donkey anti rabbit igg hrp

Manufactured by Thermo Fisher Scientific

Sourced in United States

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

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Donkey anti-rabbit–HRP IgG Rabbit

6 protocols using donkey anti rabbit igg hrp

Quantifying SP-D Binding to LPS Dotted Membranes

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LPS (O182:B12) dilutions at 2.5, 5, 10, 20, 60, and 80 μg/mL were dotted (2 μL) onto a nitrocellulose membrane (Amersham Biosciences). SP-D (370 ng) and SP-D buffer were also applied as positive and negative controls, respectively. After drying at room temperature (RT), membranes were blocked with 5% (w/v) BSA in TBST-Ca buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.02% (v/v) Tween and 5 mM CaCl₂) overnight at 4 °C. Three washes with TBST-Ca buffer were done and the different membranes were incubated with 5 μg/mL or 15 μg/mL of SP-D in TBST-Ca or 5 μg/mL of SP-D in TBST-EDTA (20 mM) for 5 h at RT. A control set with TBST-Ca without SP-D was also performed. After incubation, membranes were washed four times with TBST-Ca and incubated with SP-D antibody against human recombinant SP-D (custom polyclonal anti-SP-D generated in rabbits by Cocalico Biologicals, PA, USA)^{66 (link)} at 1 μg/mL ON at 4 °C, followed by four washes with TBST-Ca. Membranes were then incubated for 1 h with the secondary antibody, donkey anti-rabbit IgG-HRP (ThermoFisher; 1:10000) and washed as described above. SP-D complexes were detected by ECL (Millipore).

Arroyo R., Khan M.A., Echaide M., Pérez-Gil J, & Palaniyar N. (2019). SP-D attenuates LPS-induced formation of human neutrophil extracellular traps (NETs), protecting pulmonary surfactant inactivation by NETs. Communications Biology, 2, 470.

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Western Blot Analysis of HENMT1 in Testes

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Proteins were extracted from 10 week-old adult testes (n = 3/genotype) using NP-40 buffer (Fluka). 20μg of protein was separated on a 12% SDS-PAGE gel, proteins transferred to PVDF membranes and then probed using HENMT1 and actin (Sigma Aldrich) antibodies. Bound antibody was detected using donkey anti-goat IgG HRP (Dako) secondary antibody and donkey anti-rabbit IgG HRP respectively and detected with enhanced chemiluminescence (ECL Plus) detection kit (Thermo Scientific).

Lim S.L., Qu Z.P., Kortschak R.D., Lawrence D.M., Geoghegan J., Hempfling A.L., Bergmann M., Goodnow C.C., Ormandy C.J., Wong L., Mann J., Scott H.S., Jamsai D., Adelson D.L, & O’Bryan M.K. (2015). HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse. PLoS Genetics, 11(10), e1005620.

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Western Blotting of Phosphorylated Proteins

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Differentially treated cells were harvested, and 3 to 10 µg of protein per sample was used for Western blotting as described before [63 (link),97 (link)]. Used primary antibodies were anti-phospho-GSK3b (1:250, rabbit; #9336, Cell Signaling, Danvers, MA, USA), anti-phospho-Akt (1:250, rabbit; #4060, Cell Signaling), anti-phospho-mTOR (1:250, rabbit; #2971, Cell Signaling), anti-phospho-4E-BP1 (1:1000, rabbit; #2855, Cell Signaling), and anti-phospho-P70S6K (1:1000, rabbit; #9205, Cell Signaling) in 5% bovine serum albumin/tris-buffered saline with 0.1% Tween 20 (TBST). Secondary antibody was donkey-anti-rabbit IgG-HRP (1:12,500; A16035, Thermo Fisher Scientific) in 2% (w/v) casein/TBST. Loading of equal amounts of protein was confirmed by stripping and incubation of the membranes with anti-GAPDH (1:200, mouse; sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA) in 2% (w/v) casein/TBST and the secondary antibody donkey-anti-mouse IgG-HRP (1:10,000; A16011, Thermo Fisher Scientific) in 2% (w/v) casein/TBST. The signal densities were quantified using ImageJ^® software. The signals were normalized to GAPDH, and the n-fold signal induction was determined in relation to the respective control samples (control = 1).

Hellmold D., Kubelt C., Daunke T., Beckinger S., Janssen O., Hauck M., Schütt F., Adelung R., Lucius R., Haag J., Sebens S., Synowitz M, & Held-Feindt J. (2023). Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells. International Journal of Molecular Sciences, 24(10), 9075.

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Quantitative Western Blot Analysis of Retinal Proteins

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Individual dissected posterior eyecups were lysed in 160 μL of HNTG detergent buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 1% Triton X-100) freshly supplemented with 1% protease and phosphatase inhibitor cocktails (#P8340, Sigma-Millipore, and #78420, Thermofisher, respectively). Cleared lysates representing equal eyecup tissue fractions were separated on 12% SDS polyacrylamide gels using a standard Tris–glycine buffer system (Novex gels and solutions, Thermofisher). Proteins were transferred to nitrocellulose membranes (#88018, Thermofisher) for immunodetection with primary and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The primary antibodies used were porin (#4866, Cell Signaling, 1:5,000), RhoA (#sc-179, Santa Cruz Biotechnologies, Santa Cruz, CA, USA, 1:200), rhodopsin (clone B6-30, 0.01 μg/mL), and α-tubulin (#9099, Cell Signaling, 1:5,000). The secondary antibodies used were donkey-anti-rabbit IgG-HRP (#16029, Thermofisher, 1:10,000) and donkey-anti-mouse IgG-HRP (#16011, Thermofisher, 1:5,000). Chemiluminescence reaction signals from ECL-plus substrate (#NEL105001EA, Perkin-Elmer, Shelton, CT, USA) were captured with X-ray films (#3018, Denville Scientific, Swedesboro, NJ, USA), which were scanned and quantified by densitometry using Image Quant^TM TL 7.0 (GE Healthcare, Chicago, IL, USA).

Mao Y, & Finnemann S.C. (2021). Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK. Cells, 10(8), 1927.

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Antibodies for Western Blot and Immunofluorescence

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The antibodies used in this study were obtained from the following sources: anti-survivin (71G4B7, Cat#2808), anti-XIAP (3B6, Cat#2045), anti-phospho-JAK1 (Y1034/1035; Cat#3331), anti-phospho-STAT3 (Y705; D3A7, Cat#9145), anti-cleaved PARP (Cat#5625T), anti-cleaved caspase 3 (Cat#9664) and anti-phospho-JAK2 (Y1007/1008; Cat#3771) from Cell Signaling Technology Inc. (Beverly, MA, USA); anti-actin (I-19, Cat#sc-1616), anti-GAPDH (0411, Cat#sc-47724), anti-JAK1 (B-3, Cat#376996), anti-phospho-STAT3 (Y705; B-7, Cat#sc-8059), anti-STAT3 (F-2, Cat#sc-8019), from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-vinculin (Cat#V-4505) from Sigma-Aldrich. Antibody binding was detected with donkey anti-mouse IgG-HRP (Cat#A16017), donkey anti-rabbit IgG-HRP (Cat#A16029) and donkey anti-goat IgG-HRP (Cat#A15999) from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Alexa Fluor™ 488-conjugated donkey anti-mouse (Cat#A31572, Molecular Probes, Eugene, OR, USA) was used for antibody binding detection in immunofluorescence assays.

Martínez-García D., Pérez-Hernández M., Korrodi-Gregório L., Quesada R., Ramos R., Baixeras N., Pérez-Tomás R, & Soto-Cerrato V. (2019). The Natural-Based Antitumor Compound T21 Decreases Survivin Levels through Potent STAT3 Inhibition in Lung Cancer Models. Biomolecules, 9(8), 361.

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DNA Damage Response Pathway Profiling

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The following primary antibodies were used: anti-β-Actin (Thermo Fisher Scientific; #MA1-140), anti-pDNA-PKcs-S2056 (Thermo Fisher Scientific; #PA5-78130), anti-DNA-PKcs (Bethyl Laboratories; #A300-516A), anti-pRPA2-S4/8 (Bethyl Laboratories; #A300-245A), anti-RAD51 (Merck Millipore; #PC130), anti-pKAP1-S824 (Bethyl Laboratories; #A300-767A), anti-pATM-S1981 (Cell Signaling Technology; #13050S), anti-KAP1 (Bethyl Laboratories; #A300-274A), anti-pCHK1-S345 (Cell Signaling Technology; #2341), anti-CHK1 (Santa Cruz Biotechnology; #sc-8408), anti-pH2AX-S139 (Bethyl Laboratories; A300-081A), anti-BRCA1 (Cell Signaling Technology; #9010), anti-CTIP (Bethyl Laboratories; #A300-488A), anti-BLM (Bethyl Laboratories; #A300-110A), anti-V5 (Thermo Fisher Scientific; #R960-25). The secondary antibodies used were Goat anti-Mouse IgG-HRP (Thermo Fisher Scientific; #NA931V) and Donkey anti-Rabbit IgG-HRP (Thermo Fisher Scientific; #NA934V).

Dibitetto D., Sanchi A., Sanford E.J., Lopes M., & Smolka M.B. (2021). Fork Slowing and Reversal as an Adaptive Response to Chronic ATR Inhibition.

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