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Donkey anti mouse igg hrp

Manufactured by Thermo Fisher Scientific
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Donkey-anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to primary mouse antibodies in various immunoassay and immunohistochemistry applications.

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7 protocols using donkey anti mouse igg hrp

1

Western Blot Analysis of Complement Proteins

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Serum samples were incubated in RIPA buffer (Thermofisher) supplemented with protease inhibitor (Pierce). Total protein was quantified using BSA protein assay kit (Pierce). Proteins were resolved in Mini‐PROTEAN TGX gels (Bio‐Rad) and transferred to polyvinylidene membrane (Immobilion). C1q was detected with rabbit IgG anti‐human/mouse C1q (Invitrogen). Factor B was detected with rabbit IgG anti‐factor B (Invitrogen). Human IgM was detected with mouse IgG anti‐human IgM (Novusbio) with goat anti rabbit‐IgG‐HRP or donkey anti‐mouse‐IgG‐HRP (Invitrogen) as a secondary antibody. Streptavidin‐HRP (R&D systems) and crescendo western HRP substrate (Immobilon) were used prior to acquisition with Chemi‐Imager (Azure Biosystems).
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2

Quantification of Complement Proteins

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Serum samples were incubated in RIPA buffer (Thermofisher) supplemented with protease inhibitor (Pierce). Total protein was quantified using BSA protein assay kit (Pierce). Proteins were resolved in Mini-PROTEAN TGX gels (Bio-Rad) and transferred to polyvinylidene membrane (Immobilion). C1q was detected with rabbit IgG anti-human/mouse C1q (Invitrogen). Factor B was detected with rabbit IgG anti-factor B (Invitrogen). Human IgM was detected with mouse IgG anti-human IgM (Novusbio) with goat anti rabbit-IgG-HRP or donkey anti-mouse-IgG-HRP (Invitrogen) as a secondary antibody. Streptavidin-HRP (R&D systems) and crescendo western HRP substrate (Immobilon) were used prior to acquisition with Chemi-Imager (Azure Biosystems).
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3

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).
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4

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 QuantTM TL 7.0 (GE Healthcare, Chicago, IL, USA).
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5

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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6

Immunohistochemical Analysis of Placental Tissues

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Normal human placental villous tissues were collected after informed consent from elective terminations according to an IRB approved protocol at the Bao'an Maternal and Child Health Care Hospital of Shenzhen, described below. Immunohistochemistry (IHC) was performed according to our previously published procedures 21 (link). Briefly, human and mouse tissue sections (5 μm) were deparaffinized and rehydrated. Slides were then microwaved in antigen retrieval buffer (0.01 M sodium citrate, pH 6.0). After cooling, slides were incubated with hydrogen peroxide (3% w/v) for 20 min to inactive endogenous peroxidases, washed in TBS, and incubated with BSA for 30 min. Sections were then incubated with biotin-plCSA-BP (50 ng/mL), biotin-SCR (50 ng/mL), mouse monoclonal anti-C4S antibody (2B6, 1:20; Amsbio, UK), or control IgG (Abcam, UK) overnight at 4°C. Tissue sections were then washed and incubated with HRP-donkey anti-mouse IgG (1:200; Thermo Fisher Scientific, USA) or HRP-streptavidin (1:200; Boster, Wuhan, China). Hematoxylin (blue) was used as a counter-stain. Staining was visualized on a light microscope (Olympus BX53, Japan).
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7

Chondroitin Sulfate Expression in Placenta

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Immunohistochemistry was employed to evaluate CSA expression in placenta tissues. After being deparaffinized and rehydrated, slides were treated by antigen retrieval buffer. Then the tissue sections were incubated with BSA for 1 h at room temperature. Then the sections were incubated with mouse monoclonal anti-chondroitin sulfate antibody (Abcam, Cambridge, United Kingdom) or control IgG (Abcam) at 4°C overnight. After being washed, the sections were incubated with HRP-donkey anti-mouse IgG (ThermoFisher, Waltham, United States). The sections were observed under a light microscope.
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