Skin biopsies were subjected to total protein extraction, and 20 μg was used for SDS-PAGE and transferred to an immobilon-P membrane (Millipore®). The membrane was blocked with 5% nonfat dry milk in 1 TBS (20 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl, and 0.5% Tween-20) for 1 h, blotted with an anti-CD34 (1 : 2000) primary antibody overnight, and then incubated with a horseradish peroxidase- (HRP-) linked secondary antibody (GE Healthcare®) for 1 h. The membranes were developed using the Millipore Immobilon Western Chemiluminescent HRP Substrate according to the manufacturer's instructions. Chemiluminescence was detected using a FujiFilm LAS-3000 imaging system.
Horseradish peroxidase hrp linked secondary antibody
Manufactured by GE Healthcare
Sourced in United States
The Horseradish peroxidase- (HRP-) linked secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody conjugated with the enzyme horseradish peroxidase. This enzyme can catalyze the conversion of a substrate into a colored or luminescent product, allowing for the detection and quantification of target analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Non fat dry milk, by AppliChem (1 mentions)
Amersham protran premium 0.45 nc, by GE Healthcare (1 mentions)
Tbs t, by Merck Group (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Ecl plus western blotting reagent kit, by GE Healthcare (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Non fat milk, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
β mercaptoethanol, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
6 protocols using horseradish peroxidase hrp linked secondary antibody
1
Western Blot Analysis of CD34 Protein
2
Quantitative Protein Analysis via Western Blot and Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total supernatant was separated on a 12.5% SDS polyacrylamide gel, and blotted onto Amersham Protran Premium 0.45 NC (GE Healthcare Life Sciences). The membrane was blocked overnight at 4°C in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T, Sigma-Aldrich) and 5% non-fat dry milk (AppliChem). After washing with 0.05% TBS-T, the membrane was incubated with a polyclonal rabbit anti-TTR antibody (S2 Table ) diluted 1:500 and incubated for two hours at room temperature. Horseradish peroxidase (HRP)-linked secondary antibody was used (GE Healthcare Life Sciences). Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) was added. Blots were quantified using ImageJ 1.48v analysis software. TTR of cell culture supernatants was analyzed using liquid chromatography-high resolution accurate mass MS (LC-HRAM MS) in positive-ion mode.
3
Western Blot Analysis of C5AR1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins extracted from the cell samples were separated on a 4%-12% Bis-Tris NuPAGE gel and transferred onto a polyvinylidene difluoride membrane using a Trans-blot SD semidry transfer cell (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary C5AR1 antibody (1 : 100; Santa Cruz Biotechnology) overnight, followed by horseradish peroxidase- (HRP-) linked secondary antibody (1 : 5000; GE Healthcare, Piscataway, NJ, USA). Signal detection was performed with the ECL-Plus western blotting reagent kit (GE Healthcare).
4
Protein extraction and Western blot analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA buffer (Sigma‐Aldrich) supplemented with the Protease and Phosphatase Inhibitor Single‐Use Cocktail (1% (v/v), Gibco/Thermo Fisher Scientific) on ice for 15 min. Then the samples were centrifuged at 14 000g for 15 min at 4 °C to collect the supernatant. After that, soluble protein samples were denatured at 95°C with the laemmli buffer (Biorad, Hercules, CA, United States) containing 10% (v/v) β‐mercaptoethanol (Biorad). The protein samples were fractionated electrophoretically on NuPAGE™ 4%–12% Bis‐Tris Gel (Invitrogen), and then transferred to membranes by the semi‐dry (Invitrogen) or wet transfer method (BioRads). After being blocked with 3% non‐fat milk (BioRad), membrane was incubated with primary antibodies overnight at 4 °C on a shaker. Antibody information is provided in Table S2 . Following incubation with horseradish peroxidase (HRP)‐linked secondary antibodies (GE Healthcare Life Sciences, Malborough, MA, USA), the membranes were incubated with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged with ChemiDocTMTouch Imaging system (BioRad). Image J was utilized to semi‐quantify the blot images.
5
Western Blot Analysis of Signaling Pathways
6
Validating BRI2 Interactions via Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to validate BRI2 interactions, immunobloting assays were carried out. Brain lysates and eluted proteins from the co-immunoprecipitation assays performed in rat cortex tissue were resolved on 5–20% gradient SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. Moreover, additional co-immunoprecipitations were performed as described above, using a mouse anti-PSD-95 antibody (1 µg/100 µg protein; Cat. No. MAB1598; Millipore), a mouse anti-synaptophysin (5 µg/IP; Cat. No. 101011; Synaptic Systems) and a rabbit anti-GAP-43 antibody (1 µg/100 µg protein; Cat. No. 442695; Calbiochem).
Nonspecific protein-binding sites were blocked using 5% nonfat dry milk in TBST. Afterward, the membranes were incubated with the following primary antibodies: anti-BRI2 antibody (catalog no.: sc-374362, Santa Cruz Biotechnology) and anti-PSD95 (catalog no.: ab9708, Merck Millipore) overnight at 4 °C with shaking. The membranes were then washed three times for 10 minutes with TBST and incubated for 2 h at room temperature with horseradish peroxidase (HRP)-linked secondary antibodies (GE Healthcare). Finally, the membranes were washed three times for 10 minutes with TBST and enhanced chemiluminescence-based system were used for protein detection.
Nonspecific protein-binding sites were blocked using 5% nonfat dry milk in TBST. Afterward, the membranes were incubated with the following primary antibodies: anti-BRI2 antibody (catalog no.: sc-374362, Santa Cruz Biotechnology) and anti-PSD95 (catalog no.: ab9708, Merck Millipore) overnight at 4 °C with shaking. The membranes were then washed three times for 10 minutes with TBST and incubated for 2 h at room temperature with horseradish peroxidase (HRP)-linked secondary antibodies (GE Healthcare). Finally, the membranes were washed three times for 10 minutes with TBST and enhanced chemiluminescence-based system were used for protein detection.
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!