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Donkey anti goat igg hrp

Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States

Donkey anti-goat IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to goat primary antibodies in various immunoassays and detection techniques.

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6 protocols using donkey anti goat igg hrp

1

Quantifying SINE Compound Inhibition of RSV

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Overnight cultures of A549 cells were infected with RSV-A2 at multiplicity of infection (MOI) = 1 and incubated for 1 h with periodic gentle swirling. The viral inoculum was decanted and replaced with DMEM supplemented with 2% (v/v) heat inactivated FBS and 1× PSN. After 2 h of incubation, the infected cells were treated with increasing doses of SINE compounds up to 48 h.p.i. Cells treated with DMSO alone were used as control. At the end of incubation, the supernatant was decanted, and the cells were fixed with methanol + 2% H2O2 followed by air-drying overnight. The viral titre was determined using an immuno-plaque assay with a goat anti-RSV antibody (Merck Millipore, VIC, Australia) and donkey anti-goat IgG-HRP (Thermofisher, VIC, Australia). The percentage reduction in viral titre associated with SINE treatment was determined in comparison to DMSO-treated cells. The values were fitted to a non-linear regression curve using GraphPad Prism v.8.4.3 to determine the IC50, the concentration of drug required to induce 50% inhibition of viral replication.
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2

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.
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3

Western Blot Analysis of Cell Signaling

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TKE2 or HEK293 cells were homogenized in RIPA buffer (50mM Tris base, 150mM NaCl, 0.5% deoxycholic acid-sodium salt, 2% SDS, and 1% NP40, pH 7.5) containing 1x protease inhibitor cocktail (Sigma P8340). Cell lysates (20µg) from each sample were separated on a 4–20% linear gradient Tris-HCl denaturing polyacrylamide Ready Gel (Bio-Rad) and transferred to PVDF membrane (Whatman). Membranes were blocked with 5% nonfat milk in TBST (10mM Tris-HCl pH 8.0, 150mM NaCl, 0.05% Tween 20) and probed with primary antibody in the same buffer overnight at 4°C. After three washes in TBST, membranes were probed with HRP-conjugated secondary antibody for an hour at room temperature and bond second antibody was further detected using an enhanced chemiluminescence assay (Supersignal West Pico, #34080; Thermo-Fisher Scientific) and examined and photographed using a VersaDoc 4000MP imaging system (Bio-Rad). Primary antibodies, rabbit anti-Shp2 (D50F2) (cell signaling, Cat: 3397P), rabbit anti-∆Np63 (Abcam, ab166857), rabbit anti-Ngf (LSBio LS-C171793), and goat anti-β-actin (Santa Cruz, sc-1616) were used to examine expression of Shp2, ∆Np63, Ngf, and β-actin, respectively. The secondary antibodies, goat anti-rabbit IgG-HRP (Thermo-Fisher Scientific, G-21234), donkey anti-goat IgG-HRP (Thermo-Fisher Scientific, A16005) were used to assist in the detection by western blots.
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4

Western Blot Analysis of Cell Signaling

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TKE2 or HEK293 cells were homogenized in RIPA buffer (50mM Tris base, 150mM NaCl, 0.5% deoxycholic acid-sodium salt, 2% SDS, and 1% NP40, pH 7.5) containing 1x protease inhibitor cocktail (Sigma P8340). Cell lysates (20µg) from each sample were separated on a 4–20% linear gradient Tris-HCl denaturing polyacrylamide Ready Gel (Bio-Rad) and transferred to PVDF membrane (Whatman). Membranes were blocked with 5% nonfat milk in TBST (10mM Tris-HCl pH 8.0, 150mM NaCl, 0.05% Tween 20) and probed with primary antibody in the same buffer overnight at 4°C. After three washes in TBST, membranes were probed with HRP-conjugated secondary antibody for an hour at room temperature and bond second antibody was further detected using an enhanced chemiluminescence assay (Supersignal West Pico, #34080; Thermo-Fisher Scientific) and examined and photographed using a VersaDoc 4000MP imaging system (Bio-Rad). Primary antibodies, rabbit anti-Shp2 (D50F2) (cell signaling, Cat: 3397P), rabbit anti-∆Np63 (Abcam, ab166857), rabbit anti-Ngf (LSBio LS-C171793), and goat anti-β-actin (Santa Cruz, sc-1616) were used to examine expression of Shp2, ∆Np63, Ngf, and β-actin, respectively. The secondary antibodies, goat anti-rabbit IgG-HRP (Thermo-Fisher Scientific, G-21234), donkey anti-goat IgG-HRP (Thermo-Fisher Scientific, A16005) were used to assist in the detection by western blots.
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5

Molecular Mechanisms of DMF in Cellular Stress

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Hydrogen peroxide (H2O2, #MKBK8393V) and paraformaldehyde solution (#SZBC2290V) were purchased from Sigma-Aldrich. 5-Bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal, 11680293001) was purchased from Roche. Antibodies against the following proteins were used: DRG2 (Proteintech group, 14743-1-AP), acetyl-p53 (ac-p53, Cell Signaling Technology (CST), #2525), p53 (CST, #2524), p21Waf1/Cip1 (Santa Cruz Biotechnology (SCBT), sc-397), cyclin D1 (SCBT, sc-717), p16INK4α (BD Pharmingen, 551163), SIRT1 (Merck Millipore, 07-131), SIRT1 (Abcam, ab110304), acetyl-NF-κB p65 (ac-NF-κB p65, CST, #3045), NF-κB p65 (SCBT, sc-8008), phosho-Histone H2A.X (p-H2A.X, CST, 9718 s), and β-actin (SCBT, sc-1616). Secondary antibodies for immunoblotting were from SCBT (goat anti-mouse IgG-HRP, sc-2005; mouse anti-rabbit IgG-HRP, sc-2357; donkey anti-goat IgG-HRP, sc-2020), and those for immunofluorescent staining were from Invitrogen (goat anti-rabbit Alexa Fluor 568, A-11011; goat anti-mouse Alexa Fluor 488, A-11001; goat anti-mouse Alexa Fluor 568, A-11004). DMF (6,4′-dihydroxy-7-methoxyflavanone) was obtained from the Standardized Material Bank for New Botanical Drugs (no. NNMBP012), Wonkwang University (Republic of Korea). DMF (>98%) was isolated from the heartwood of Dalbergia odorifera [25 (link)] and was dissolved in DMSO (0.05% in final culture concentration).
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6

OMGP Enrichment and Detection

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CSF samples were pooled and approximately 30 × concentrated using filter columns (Amicon 3 kDa). These, together with recombinant OMGP and immunoglobulins were separated by SDS gel (Invitrogen, NP0321) and transferred on an activated (10% methanol) PVDF membrane (GE Healthcare, 10600023). As primary Ab, goat anti-OMGP (R&D, AF1674) was used. As secondary Ab, donkey anti-goat-IgG-HRP (Invitrogen, A16005) was used, because it shows only minimal cross-reactivity with human IgG due to absorption against human IgG. ECL prime solution (GE Healthcare, RPN2232) was used and signal was detected by digital imaging systems Odyssey Fc from Leica.
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