The largest database of trusted experimental protocols

Hrp coupled secondary antibody

Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom

HRP-coupled secondary antibodies are a type of labeling reagent used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. These antibodies are conjugated with the enzyme Horseradish Peroxidase (HRP), which is used as a detection system to visualize target antigens. HRP-coupled secondary antibodies bind to the primary antibody that recognizes the target antigen, and the presence of the target can be detected through the enzymatic activity of HRP, typically using a colorimetric or chemiluminescent substrate.

Automatically generated - may contain errors

35 protocols using hrp coupled secondary antibody

1

Western Blot Analysis of Lipid Metabolism Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Tissue samples were lysed in RIPA buffer supplemented with complete mini protease inhibitor cocktail (Roche) and 50 mM Na3VO4 using a TissueLyzer (Qiagen). 20 µg protein was separated on 10% Tris–glycine gels. Transfer to a nitrocellulose membrane (GE) was performed in a wet blotting system for 2 h at 400 mA. Membranes were then blocked for 1 h in 5% milk in TBS-Tween (TBST) and after rinsing with TBST, membranes were incubated in the respective primary antibody overnight at 4 °C. Primary antibodies and dilutions were: CD36 (Novus Biologicals, 1:1000); LRP1 (Abgent, 1:25,000); LPL (Kind gift from Stefan K. Nilsson, 1:1000), SCD1 (Santa Cruz, 1:250), LPL (Cell Signaling, 1:1000). All primary antibodies were diluted in 5% BSA in TBS-T. After incubation, the membranes were washed 3 times 10 min in TBST and incubated for 1 h with HRP-coupled secondary antibodies (all from Jackson, all 1:5000 in 5% milk in TBS-T). After 3 additional washes in TBST, detection was performed using Amersham ECL Prime Western Blotting Detection Reagent (GE) and Amersham Hyperfilm (GE) autoradiography films. The films were developed using Kodak Developer and Fixer.
+ Open protocol
+ Expand
2

Protein Extraction and Western Blotting for Worms

Check if the same lab product or an alternative is used in the 5 most similar protocols
For Western blotting experiments, we collected individual worms by hand and boiled them in Laemmli protein sample loading buffer prior to gel separation. Specific proteins were analyzed by western blotting with ECL detection (GE Healthcare) of HRP-coupled secondary antibodies (Jackson ImmunoResearch). Worm protein extracts for sucrose gradients and ribosome immunoprecipitations were made as described (5 (link)). The sucrose gradient centrifugation and fractionation was conducted as previously described (10 (link)).
+ Open protocol
+ Expand
3

Western Blot Analysis of Protein Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells on a thermo-shaker were lysed for 10 min at 95 °C in Laemmli buffer. Samples were size separated via SDS-PAGE. Proteins were transferred onto nitrocellulose membranes with Pierce™ Power blotter (Thermo Fisher Scientific, Waltham, MA, USA). The following antibody dilutions were used: Syn5 (1:1000), GS27 (1:1000), GS28 (1:1000), and GFP (1:5000). Corresponding HRP-coupled secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used. Protein bands were visualized with SuperSignal (Thermo Scientific), and analyzed on a Fusion S western blot imager (Vilber Lourmat, Marne-la-Vallée, France). Densitometry analysis of the gels was carried out using ImageJ software from the NIH.
+ Open protocol
+ Expand
4

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were lysed for 15 min at 4 °C in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P40) supplemented with protease inhibitors (CompleteTM protease inhibitor mixture, Roche, Rotkreuz, Switzerland). Supernatants were collected, and the protein concentration was determined with a BCA assay (Sigma, Deisenhofen, Germany). Equal amounts of protein samples were separated on 8% Tris/glycine gels and then transferred to nitrocellulose membranes (GE Healthcare, Uppsala, Sweden). Subsequently, the membranes were incubated with primary antibodies and HRP-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania, USA). Chemiluminescence was measured, using an imager and the software Fusion (Vilber Loumat).
+ Open protocol
+ Expand
5

Plasma Membrane Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Three confluent 160-mm-diameter dishes of MBECs were harvested and plasma membranes were isolated as previously described (Yao et al., 2009). Protein concentrations were determined by Bio-Rad Dc-Protein assay (Bio-Rad). Samples were mixed with Bio-Rad XT sample buffer and reducing agent, run on Criterion™ XT 4–12% Bis-Tris Gels (Bio-Rad), and transferred onto PVDF membranes. Immunodetection was performed with (1:100) rabbit anti-connexin 43 antibody (Invitrogen), (1:500) mouse anti phospho-caveolin1 (pY14) (BD Transduction Laboratories), (1:5000) rabbit anti-β-catenin (Abcam), (1:1000) rabbit anti-Cav-1 (Abcam), or (1:1000) mouse-anti-transferrin receptor (Life Technology) followed by HRP-coupled secondary antibodies (Jackson ImmunoResearch), and developed with ECL-Plus (GE Healthcare). Protein bands were quantified by densitometry using Image Lab 5.1 (Bio-Rad) software. Plasma membrane connexin43 levels were normalized to plasma membrane transferrin receptor. This value was normalized to total β-catenin as a housekeeping reference protein.
+ Open protocol
+ Expand
6

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
Samples were subjected to SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat dry milk in TBST (50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween-20) and incubated with primary antibodies as indicated. Antibodies used were: mouse anti-c-Myc 9E10 (1:1000, Thermo Fisher Scientific, Waltham, MA), mouse anti-FLAG (1:1000, Sigma Aldrich, St. Louis, MI) and mouse anti-actin (1:2000, Merck Millipore, Burlington, MA). Primary antibodies were detected using HRP coupled secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, Cambridge House, UK). Chemiluminescent signal was developed using Clarity and ClarityMax Western Blotting Substrates (BioRad Laboratories Inc., Hercules, CA) followed by detection with a FujiFilm ImageQuant LAS-4000 detector (GE Healthcare, Chicago, IL).
+ Open protocol
+ Expand
7

Quantifying Protein Expression via Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols
Protein concentration was measured using the bicinchoninic acid (BCA) method (Thermo Scientific). Subsequently, 20 μg of cell lysate was denatured with sample buffer (62.5 mM Tris–HCl pH6.8, 2% (v/v) SDS, 50 mM DTT, 10% (v/v) glycerol), subjected to 4%–20% gradient SDS-PAGE, and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked for 1 h with 5% (w/v) dried skimmed milk in TBS-T buffer (25 mM Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20, pH 7.4), and incubated with the following primary antibodies: mouse anti-GFAP (Sigma-Aldrich; 1:50,000), rabbit anti-Iba1 (Wako; 1:10,000), mouse anti-β-actin (Sigma-Aldrich; 1:20,000), anti-NSE (1:1000, Dako), or anti-GAPDH (1:40000, GeneTex), at 4°C overnight. Then, membranes were incubated with the corresponding HRP-coupled secondary antibodies (Jackson Immunoresearch; 1:10,000) for 1 h at room temperature, and the bands were visualized using the Super Signal West Pico chemiluminescent substrate (Thermo Scientific). The immunoreactive signals were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Iba1 and GFAP values were normalized to β-ACTIN values. NSE results, due to molecular weight, were normalized to GAPDH values.
+ Open protocol
+ Expand
8

Whole-Embryo Protein Extraction and Western Blotting

Check if the same lab product or an alternative is used in the 5 most similar protocols
To obtain whole-embryo protein lysate, embryos were sonicated in fresh buffer (10 mM Hepes, pH 7.5, 100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, pH 8.0, 1 mM NaF, 1 mM Na3VO4, 0.5% Triton X-100, and protease inhibitor cocktail tablets [1 tablet/10 ml; Roche]). Lysate was cleared by centrifugation, and supernatant was collected and stored after addition of 5× SDS sample buffer (10% SDS, 20% glycerol, 0.2 M Tris-HCl, pH 6.8, 0.05% Bromophenol blue, and 10% β-mercaptoethanol added right before use). Prepared protein samples were separated by SDS-PAGE with the Mini-PROTEAN vertical electrophoresis cell system (Bio-Rad Laboratories), transferred to a polyvinylidene difluoride membrane (Immobilon-P) in a semidry transfer cell (Bio-Rad Laboratories), probed with anti-ASC (1:103 or 1:104 dilution) or anti-GFP (1:104 dilution; Santa Cruz Biotechnology, Inc.), and developed with the corresponding HRP-coupled secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Detection was performed using Luminata Crescendo Western HRP substrate (EMD Millipore).
+ Open protocol
+ Expand
9

In-gel activity assay for complex V

Check if the same lab product or an alternative is used in the 5 most similar protocols
In-gel activity of complex V was determined as previously described39 (link). In brief, a gel strip was cut from the BN-PAGE gel and incubated for 30 min in complex V calibration buffer (35 mM Tris, 220 mM glycine pH 8.3). After this, the gel was transferred to complex V staining buffer (35 mM Tris, 220 mM glycine pH 8.3, 14 mM MgSO4, 0.2% (w/v) Pb(NO3)2, 8 mM ATP) and incubated at 30 °C until bands appeared. TCA protein precipitation was performed according to standard procedure. SDS-PAGE, urea-PAGE, and western blotting were performed according to standard protocols. Signals were detected using HRP-coupled secondary antibodies (used in 1:10,000 dilution, Jackson ImmunoResearch, code number 111-035-144) and enhanced chemiluminescence system (GE Healthcare). No other image processing, other than cropping, scaling, and contrast adjustment using Adobe Photoshop CS6 and Adobe Illustrator CS6 was applied.
+ Open protocol
+ Expand
10

Immunoblotting Analysis of β-catenin and nAChR

Check if the same lab product or an alternative is used in the 5 most similar protocols
Dissected LAL muscles were homogenized in RIPA buffer (150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) containing a cocktail of protease inhibitors (Halt™ Protease Inhibitor Cocktail 100X; #78430, Thermo ScientificTM) along with 2 mM Na3VO4. For immunoblotting, proteins were separated in 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Scientific), and probed with rabbit anti β-catenin (1:1000, Millipore), rabbit anti nAChR γ-subunit (1:1000, Invitrogen), or mouse anti GAPDH (1:1000; Santa Cruz Biotechnologies). Bound antibodies were visualized using the respective HRP-coupled secondary antibodies (Jackson ImmunoResearch Laboratories) developed using a chemiluminescence reagent kit (Perkin Elmer).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!