Proteins (30–100 µg) were extracted and analysed by western blotting as described53 (link). Primary antibodies used in this study were: anti-RBM14 (Euromedex, ref. 10196-1-AP), anti-PPARγ (Santa Cruz, ref. sc-7196), anti-TFIIB (Santa Cruz, ref. sc-225), anti-β-actin (Santa Cruz, ref. sc-1616), anti-H3 (Abcam, ref. ab1791) and control IgG (Merck-Millipore, ref. 17–658). Secondary antibodies were anti-rabbit IgG-peroxidase antibody (Sigma, ref. A0545) or anti-goat IgG-peroxidase antibody (Sigma, ref. A5420).
Anti rabbit igg peroxidase antibody
Manufactured by Merck Group
Sourced in United States
Anti-rabbit IgG-peroxidase antibody is a laboratory reagent used for the detection and quantification of rabbit immunoglobulin G (IgG) in various immunoassays. It is a conjugate of an anti-rabbit IgG antibody and the enzyme peroxidase, which can catalyze a colorimetric reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg peroxidase antibody, by Merck Group (6 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pierce ecl reagent, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti tfiib, by Santa Cruz Biotechnology (1 mentions)
Control igg, by Merck Group (1 mentions)
Anti goat igg peroxidase antibody, by Merck Group (1 mentions)
Anti h3, by Abcam (1 mentions)
Anykd criterion tgx gels, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Seeblue plus2, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris precast polyacrylamide gels, by Thermo Fisher Scientific (1 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti ha tag, by Cell Signaling Technology (1 mentions)
Alexa fluor 568 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
21 protocols using anti rabbit igg peroxidase antibody
1
Western Blotting of Cellular Proteins
2
Antibody Production and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GyrA and Topo I proteins were purified as previously described (7 (link),37 (link)). Polyclonal rabbit antibodies against GyrA were obtained after three subcutaneous injections at 3-week intervals of polyacrylamide portions containing a 50 kDa Nterminal-GyrA fragment. Blood was recovered and the serum stored at −80°C. Polyclonal antibodies against Topo I were obtained from Davids Biotechnologie and polyclonal antibodies against LytA were kindly provided by Ernesto García (CIB, CSIC, Madrid, Spain). Whole cell lysates were separated on Any KD™ Criterion TGX gels (Bio-Rad) and transferred to PVDF membranes with Trans-Blot Turbo (Bio-Rad) at 25 V, 1 A for 30 min. The protein-transferred membranes were blocked with Super Block T20 (TBS) blocking buffer (Thermo Scientific) for 1 h and probed with antibodies anti-GyrA (diluted 1:2000), anti-Topo I (diluted 1:2000) and anti-LytA (diluted 1:20000) in Super Block for 2 h. The anti-GyrA and anti-Topo I antibodies were previously purified by affinity using purified proteins bound to a PVDF membrane. Anti-rabbit IgG-Peroxidase antibody (Sigma-Aldrich) served as the secondary antibody. Super Signal West Pico chemiluminescent substrate (Thermo Scientific) was utilized to develop the signal and monitored with a ChemiDoc™ MP system (Bio-Rad). Image analysis was performed with Image Lab™ software (Bio-Rad).
3
Gut Mucus IgT ELISA Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Direct and indirect ELISAs were performed to analyze the total and hemocyanin-specific gut mucus IgT levels, respectively, as previously described (46 (link)). The specificity of the customized polyclonal anti-GSBIgT antibody used in this study has been previously validated by Western blot and ELISA (46 (link)). Briefly, to assess the total IgT here, 96-well plates were coated with fish gut mucus diluted 1:6 in carbonate/bicarbonate solution and incubated overnight at 4°C. Similarly, to measure the hemocyanin-specific IgT level, 1:5 dilutions of fish gut mucus from the naive, hemocyanin-immunized, or pre-immune animals as control were added to a hemocyanin precoated flat-bottomed ELISA plates. Then, each assay was followed by the addition of a customized polyclonal anti-seabream IgT (GeneScript) and an anti-rabbit IgG peroxidase antibody produced in goat (Sigma-Aldrich, Spain), both diluted at 1:1,000. Finally, the chromogen TMB (Sigma-Aldrich) was added, and the plates were incubated at 22°C. The reaction was stopped with the addition of 50 μl/well of 2 M H2SO4, and the absorbance was read at 450 nm on a SPECTROstart nano (BGM; LabTechnologies). The plotted values resulted from subtracting the absorbance from the control wells.
4
Immunoblotting Assay for Rat Liver AQP8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aliquots (10 μg of total proteins) of rat liver mitochondria prepared as described above were heated to 90 °C for 5 min and electrophoresed into 10% NuPAGE Bis-Tris precast polyacrylamide gels (Invitrogen, Waltham, MA, USA) using a SeeBlue™ Plus2 prestained protein ladder (Invitrogen). The resolved proteins were submitted to immunoblotting and incubated overnight with affinity-purified rabbit antibodies against an N-terminal peptide of rat AQP8 at a final concentration of 1 μg/mL blocking solution (20 mM Tris–HCl, 0.15 M NaCl, 1% Triton X-100, pH 7.5). Horseradish peroxidase antirabbit IgG-treated membranes (antirabbit IgG peroxidase antibody; Sigma) were developed by luminal chemiluminescence (ECL WEST FEMTO PLUS, Immunological Sciences, Rome, Italy). The immunoreactive bands were analyzed by densitometry using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The density of each band was normalized against that of the housekeeper gene β-actin.
5
Western Blot and Immunofluorescence Antibody Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies for Western blot analysis: rabbit monoclonal Anti-HA-tag (Cell Signaling Technology, at 1:1,000 dilution), Rabbit monoclonal anti-SYVN1 (Cell Signaling Technology, at 1:1,000 dilution), mouse monoclonal anti-GAPDH (Abcam, at 1:2,500 dilution) rabbit monoclonal Anti-HA (Cell Signaling, at 1:1,000 dilution), mouse monoclonal anti-RFP (Thermo Fisher Scientific, at 1:1,000 dilution), rabbit anti- Endoglin P3D1 (Santa Cruz Biotechnology, at 1:200 dilution), anti-Mouse IgG Peroxidase antibody (Sigma Aldrich, at 1:40,000 dilution), and anti-Rabbit IgG Peroxidase antibody (Sigma Aldrich, at 1:30,000 dilution).
Antibodies for immunofluorescence: mouse monoclonal anti-HA-tag (Cell signaling Technology, at 1: 200 dilution), rabbit polyclonal anti-calnexin (Santa Cruz Biotechnology, at 1 : 200 dilution), rabbit anti-Histone-H3 (Cell Signaling Technology, at 1:1,000 dilution), mouse monoclonal anti-HA (Cell Signaling Technology, at 1: 200 dilution), Alexa Fluor 568-goat anti-mouse IgG (Molecular Probes, at1:200 dilution), and Alexa Fluor 488-goat anti rabbit IgG (Molecular Probes, at 1:200dilution).
Antibodies for immunofluorescence: mouse monoclonal anti-HA-tag (Cell signaling Technology, at 1: 200 dilution), rabbit polyclonal anti-calnexin (Santa Cruz Biotechnology, at 1 : 200 dilution), rabbit anti-Histone-H3 (Cell Signaling Technology, at 1:1,000 dilution), mouse monoclonal anti-HA (Cell Signaling Technology, at 1: 200 dilution), Alexa Fluor 568-goat anti-mouse IgG (Molecular Probes, at1:200 dilution), and Alexa Fluor 488-goat anti rabbit IgG (Molecular Probes, at 1:200dilution).
6
Evaluating eIF4G Expression in RRL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from mock or L-protease-treated RRL extracts were separated by a 7.5% SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes (Boehringer Mannheim, Ingelheim am Rhein, Germany). Blots were incubated first with antibody against eIF4G (#2617 Cell Signaling Technology, Danvers, MA, USA) for 16 h at 4 °C, then with an anti-Rabbit IgG–Peroxidase antibody (Sigma Aldrich, Saint-Louis, MO, USA) for 2 h. The chemiluminescence signal was detected using an Pierce ™ ECL reagent (Thermofisher, Waltham, MA, USA) in the Chemidoc Imager (Biorad, Hercules, CA, USA).
7
Whole Cell Lysate Western Blot of CRISPR Knockout
8
Western Blot Analysis of RIOK3 and TRA2-β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (10mM Tris-HCl pH 8.0, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Lysates were clarified and subsequently separated by SDS-PAGE on 10% polyacrylamide and wet transferred to PVDF. The membrane was blocked with 5% milk solution in Tris-buffered saline Tween 20 (TBST) at room temperature, and primary antibody was added at a dilution of 1:1000 in milk buffer. Secondary antibody was added at a dilution of 1:10,000 in milk buffer. Following each antibody incubation, the membrane was triple rinsed with TBST. Chemiluminescent visualization of blots was carried out using visualization solution made up of two buffers (buffer 1: 2.5 mM luminol, 0.396 mM coumaric acid, and 100 mM Tris-HCl pH 8.5; buffer 2: 0.0192% hydrogen peroxide, 100 mM Tris-HCl pH 8.5) mixed immediately before visualization.
The following primary antibodies were used: GAPDH loading control antibody MA5-15738 (Thermo Fisher Scientific), anti-RIOK3 SAB1406721 (Sigma), and anti-TRA2-β antibody ab31353 (Abcam). HRP-conjugated secondary antibodies used were anti-Mouse IgG peroxidase antibody produced in goat A2554 (Sigma) and anti-Rabbit IgG peroxidase antibody produced in goat A0545 (Sigma).
The following primary antibodies were used: GAPDH loading control antibody MA5-15738 (Thermo Fisher Scientific), anti-RIOK3 SAB1406721 (Sigma), and anti-TRA2-β antibody ab31353 (Abcam). HRP-conjugated secondary antibodies used were anti-Mouse IgG peroxidase antibody produced in goat A2554 (Sigma) and anti-Rabbit IgG peroxidase antibody produced in goat A0545 (Sigma).
9
Histopathological and IHC Analysis of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were subjected to histopathological and IHC examination as previously described [21 (link), 22 (link)]. Briefly, tissue samples were fixed in 10% neural buffered formalin for 48 h, embedded in paraffin wax and sectioned. Thin sections with 4-μm thickness stained with hematoxylin and eosin (H–E staining) for histopathological examinations. 4-μm sections were treated with 3% hydrogen peroxide in PBS for 20 min followed by washes in PBS and digestion with 0.05% protease (protease XIV; Sigma) for 5 min at 37 °C. Then washes with PBS, sections were incubated in blocking solutions with 8% skim milk for 40 min at room temperature. After washes with PBS, sections were incubated for 2 h at 4 °C with rabbit antiserum against CSFV (saved by our laboratory) diluted 1:200 in PBS. Then washes with PBS, sections were incubated with anti-rabbit IgG-peroxidase antibody produced in goat (Sigma) diluted 1:400 in PBS for 40 min at room temperature.
10
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using a reported procedure (Hervas‐Aguilar & Penalva, 2010 (link); López‐Berges et al., 2016 (link)) involving solubilization from lyophilized mycelial biomass with NaOH, followed by precipitation with trichloroacetic acid (TCA). Aliquots were resolved in 10%–12% SDS‐polycrylamide gels and transferred to nitrocellulose membranes with a Trans‐Blot Turbo transfer system (Bio‐Rad) for blotting. Western blots were reacted with Phospho‐(Ser/Thr) Akt substrate antibody (1:10,000; #9611 Cell Signalling Technology) as primary and with anti‐rabbit IgG‐peroxidase antibody (1:10,000; A1949; Sigma‐Aldrich) as secondary, or with anti‐α‐tubulin antibody (1:10,000; T6119; Sigma‐Aldrich) as primary and anti‐mouse IgG‐peroxidase antibody (1:10,000; A4416; Sigma‐Aldrich) as secondary. Proteins were detected with ECL (Amersham Biosciences).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!