Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche, 000000011836170001) on ice for 15 min. The cell lysates were rotated at 4 °C for 30 min and the insoluble material was removed by centrifugation at 15,000 g for 15 min. Protein concentrations were determined by Bio-Rad Protein Assay using bovine serum albumin (BSA) as the standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by SDS PAGE on stain-free TGX gradient gels (Bio-Rad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 hours), followed by immunoblotting with the primary antibody specified for each experiment for PIWIL2 (Abcam, ab181340 diluted at 1:1000), PIWIL4 (Abcam ab 111714, diluted at 1:1000), and beta Actin (Abcam ab1801, diluted at 1:1000). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H + L) HRP-conjugated secondary antibodies (Bio-Rad) and detected using ECL (Amresco). Densitometry was performed using Image Lab software v. 4.1 (Bio-Rad).
Goat anti rabbit igg h l hrp conjugated secondary antibody
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
Goat anti-Rabbit IgG H + L HRP-conjugated secondary antibody is a laboratory reagent that binds to and labels rabbit primary antibodies. The HRP (Horseradish Peroxidase) conjugate allows for the detection of the bound primary antibody in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab111714, by Abcam (1 mentions)
Ab1801, by Abcam (1 mentions)
Ab181340, by Abcam (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Image lab software v 4, by Bio-Rad (1 mentions)
Stain free tgx gradient gels, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Avantor (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Complete mini, by Roche (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Protease and phosphatase inhibitors cocktail, by Cell Signaling Technology (1 mentions)
Rabbit anti human n cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Supersignal west pico chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch image system, by Bio-Rad (1 mentions)
5 protocols using goat anti rabbit igg h l hrp conjugated secondary antibody
1
Western Blot Analysis of PIWIL Proteins
2
Quantitative Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins cells were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitor. The protein concentration was assessed using the bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Equal amounts of protein (20 µg/lane) were separated by 15% SDS-PAGE and transferred onto the PVDF membrane (EMD Millipore). After blocking with 5% non-fat milk for 1 h at room temperature, the membrane was probed with primary antibodies targeting JAG1 (1:1,000; cat. no. ab109536; Abcam) or GAPDH (1:3,000; cat. no. ab8245; Abcam) at 4˚C overnight. The membrane was washed three times with PBS-Tween-20 (0.1%) and then incubated with Goat anti-Mouse IgG (H+L)-horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; cat. no. 170-6516; Bio-Rad Laboratories, Inc.) or Goat anti-Rabbit IgG (H+L)-HRP-conjugated secondary antibodies (1:5,000; cat. no. 170-6515; Bio-Rad Laboratories, Inc.) at room temperature for 1 h. Following an extensive wash with PBST, the enhanced chemiluminescence western blotting kit (Pierce; Thermo Fisher Scientific, Inc.) was used to visualize the bands. Densitometric analysis was performed using ImageJ software (version 1.8.0; National Institutes of Health). The expression of GAPDH was used as the loading control.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extraction was performed by homogenizing cells lysis buffer containing 1× protease and phosphatase inhibitors cocktail (lysis buffer 10×; Cell Signaling Technology, Danvers, MA, USA). After sonication, the homogenates were centrifuged at 300 g for 5 min at 4 °C. Protein concentrations were determined using BCA (bicinchoninic acid assay) to measure the concentration of protein in a solution. Lysates obtained from 2 subpopulations were analyzed in denaturing condition by SDS-PAGE and transferred on to nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK). For the experiments with CD44v8-10-knocked-down PC3 cells, the total lysates were prepared 72 h after siRNA/scramble transfection. Membranes were incubated with primary antibodies rabbit anti-human-phospho-β catenin (Ser 33/37 Thr 41) and rabbit anti-human total β catenin, rabbit anti-human N-cadherin, rabbit anti-human P-PARγ, and rabbit anti-human RUNX2 (all from Cell Signaling Technology), and rabbit anti-human MMP-13 (Abcam, Cambridge, UK) followed by goat anti-Rabbit IgG H + L HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) and visualized by ECL (Western nova 2.0; Cyanagen, Bologna, Italy) using Chemidoc Gel Imaging System Image Lab version 5.2.1. software (Bio-Rad). Densitometric analysis of immunoblots was performed by ImageJ.
4
Quantification of Protein Expression and Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration was determined using the BCA assay kit (Beyotime). The samples were boiled at 100°C for 5 min in 5× sample buffer. The protein extracts were electrophoresed in 7.5–10% SDS polyacrylamide gels (Bio-Rad Laboratories) according to the Laemmli method [22 (link)]. The separated proteins were then transferred on to a nitrocellulose membrane in Tris–glycine buffer containing 20% methanol. The membranes were blocked and immunoblotted with a 1:1000 dilution of a primary antibody including anti-puromycin (keraFAST), anti-P-mTOR (Ser2448), anti-mTOR, anti-P-p70S6K (Thr389), anti-p70S6K, anti-P-4EBP1 (Thr37/46), anti-4EBP1, anti-P-AMPK (Thr172) and anti-AMPK (Beverly, MA, USA).
The proteins were detected using either goat anti-rabbit IgG (H+L)-HRP conjugated secondary antibody (1:2000, Bio-Rad Laboratories) or HRP-labelled goat anti-mouse IgG (H+L) secondary antibody (1:1000, Beyotime) with enhanced chemiluminescence (ECL) plus western blot detection reagents (Beyotime). β-Actin was used as an internal control (Beyotime). Western blots were developed and quantified using BioSpectrum 810 with VisionWorksLS 7.1 software (UVP LLC). The protein level was quantified by normalizing total proteins with β-Actin, and by normalizing phosphorylated proteins with their total pairs.
The proteins were detected using either goat anti-rabbit IgG (H+L)-HRP conjugated secondary antibody (1:2000, Bio-Rad Laboratories) or HRP-labelled goat anti-mouse IgG (H+L) secondary antibody (1:1000, Beyotime) with enhanced chemiluminescence (ECL) plus western blot detection reagents (Beyotime). β-Actin was used as an internal control (Beyotime). Western blots were developed and quantified using BioSpectrum 810 with VisionWorksLS 7.1 software (UVP LLC). The protein level was quantified by normalizing total proteins with β-Actin, and by normalizing phosphorylated proteins with their total pairs.
5
Quantifying Phosphorylated cPLA2 in CCI Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The whole cell lysates from sciatic nerves of CCI mice 7 days after surgery were prepared with RIPA buffer (10 mM Tris-HCL pH 7.4, 30 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, supplemented with 1 mM Na3VO4, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin and 1 mM PMSF). Protein concentration was determined for each sample, and equal amount of proteins were run on 4–15% SDS-PAGE. Thereafter, proteins were transferred onto nitrocellulose membranes, blocked for 1 h at RT with 5% non-fat milk, and incubated overnight at 4 °C with the primary rabbit antibodies against phosphorylated cPLA2 (1:300; Cat# 2831, Cell Signaling) and total cPLA2 (1:1000; Cat# 2832, Cell Signaling). After probing with goat anti-rabbit IgG (H + L)-HRP-conjugated secondary antibody (1:2500; Bio-Rad, Hercules, CA) for 1 h at 25 °C, protein bands were detected using the Supersignal West Pico Chemiluminescence (Thermo Fisher, Waltham, MA). Membranes were subsequently probed for β-actin expression to serve as a loading control. Images were acquired and analyzed with a Chemidoc Touch image system (BioRad).
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!