For protein extraction, the hearts were dissected and homogenized with CelLytic MT cell lysis reagent (Sigma‐Aldrich), followed by sonication and centrifugation. The protein concentrations were determined by protein assay (Bio‐Rad) and equal amounts (50 μg) of extracted proteins were loaded for analysis. The samples were resolved by 8% SDS‐PAGE and wet‐transferred to a polyvinylidene difluoride membrane overnight. The proteins were detected by immunoblotting using mouse monoclonal antibody anti‐Actin (Merck Millipore) and rabbit polyclonal antibody anti‐SCN5A (Aviva Systems Biology) as well as horseradish peroxidase–conjugated anti‐mouse or anti‐rabbit secondary antibody (Thermo Fisher Scientific) followed by chemiluminescent detection with Immobilon Western HRP Substrate (Merck Millipore).
Horseradish peroxidase conjugated anti mouse or anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in Sweden
Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies are laboratory reagents used for detection and quantification of target proteins in various immunoassay techniques. They are designed to bind to and amplify the signal from primary antibodies directed against mouse or rabbit proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Cellytic mt cell lysis reagent, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti alpha smooth muscle actin, by Agilent Technologies (1 mentions)
Anti β tubulin, by Fujifilm (1 mentions)
Imagequant las 4000 mini imager, by GE Healthcare (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ab97959, by Abcam (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico ecl solution, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Merck Group (1 mentions)
Hybond ecl nitrocellulose membrane, by Cytiva (1 mentions)
Fuji las 3000 system, by Fujifilm (1 mentions)
Ab41902, by Abcam (1 mentions)
Fibronectin, by Merck Group (1 mentions)
6 protocols using horseradish peroxidase conjugated anti mouse or anti rabbit secondary antibody
1
Protein Extraction and Detection
2
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as published69 (link). Briefly, we collected cell lysates in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) after treatment. Protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol with bovine serum albumin as a standard. Protein extracts were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). Then we immersed the membranes in a solution containing primary antibodies overnight at 4 °C. The primary antibodies were anti-alpha smooth muscle actin (1:1000; DAKO) and anti-β-tubulin (1:1000; Wako Pure Chemical Industries, Osaka, Japan). After washing the membranes, we incubated them in a solution with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:2000–1:5000; Thermo Fisher Scientific) for 1 h at room temperature. The proteins in the membranes were made to react with ECL substrate (Thermo Fisher Scientific), and we placed the membranes in an ImageQuant LAS 4000 mini-imager (GE Healthcare, Chicago, IL, USA) to visualize the bands and quantify them using ImageJ software (ver. 1.49, NIH, Bethesda, MD, USA).
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
The cells were harvested by centrifugation, and total protein was extracted with the Whole-Protein Extraction Solution (M-PER; Thermo Fisher Scientific Inc.) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc.). The mixture was centrifuged at 16,000 × g for 10 min, and the protein in the supernatant was subjected to quantification with the BCA Kit (Sigma-Aldrich Co. LLC). Protein samples (20 μg) were loaded onto an SDS-polyacrylamide gel; transferred to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA); blocked with 5% nonfat dried milk; and immunoblotted at 4°C overnight with primary antibodies against OCT4A (2890, Cell Signaling Technology), SOX2 (ab97959, Abcam), KLF4 (ABS1514, Merck), and α-tubulin (AbC-2001, AbClon). The next day, membranes were washed with PBST; incubated with horseradish peroxidase–conjugated anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher Scientific Inc.); treated with a SuperSignal West Pico ECL solution (Thermo Fisher Scientific Inc.); and visualized on a Fuji LAS-3000 system (Fujifilm, Tokyo, Japan). The α-tubulin served as a loading control.
4
Immunoblot Analysis of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CAFs (2 × 104 cells per 12-well dish), Hep3b and SNU423 (5 × 105 cells per 60 mm dish) and SNU398, SNU449, HLF, Huh7 and PLC/PRF5 (4 × 105 cells per 60 mm dish), after the specified treatments, were washed in ice-cold phosphate buffer saline (PBS), pH 7.4, lysed and analyzed by immunoblot as described20 (link), with antibodies at the following dilutions: fibronectin, 1:10,000 (Sigma-Aldrich, Stockholm, Sweden, F3648); fatty acid synthase (FASN), 1:1,000 (ab22759); calponin, 1:2,000 (EP798Y, ab46794); LXRα, 1:1,000 (ab41902); Smad3, 1:1,500 (ab40854), all from Abcam, Cambridge, United Kingdom; α-smooth muscle actin (αSMA), 1:500 (Santa Cruz Biotech Inc., Santa Cruz, CA, USA, sc1a4); GAPDH, 1:50,000 (Ambion, ThermoFisher Scientific, Fyrislund, Sweden, AM4300). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (ThermoFisher Scientific, Fyrislund, Sweden) were used at 1:20,000 dilution. Triplicate (nb = 3) biological experiments were performed in 2 technical replicates (nt = 2) per condition. Densitometric quantification of protein bands was performed using the Fujifilm Intelligent Dark Box II program of a Fuji Aida digital scanner (Fujifilm Nordic AB, Stockholm, Sweden).
5
Western Blot Analysis of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 and U343 cells were harvested 48 h after transfection and lysed in RIPA buffer (50-mM Tris-HCl pH 8.0, 150-mM NaCl, 2-mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1% Nonidet P-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS)) for 30 min on ice and pelleted. Protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Proteins (40–60 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (8–10%) and transferred onto Immun-BlotTM polyvinylidene difluoride membranes (Bio-Rad), and blocked with 3% bovine serum albumin (BSA) (Sigma-Aldrich). The membranes were then incubated overnight at 4°C with primary antibody, and protein bands were detected using horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher Scientific) and visualization using ECL solution (Thermo Fisher Scientific).
6
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 cellular proteins were extracted in lysis buffer (20 mM Tris-HCl, pH 7.5, 1% nonidet P‐40, 150 mM NaCl) supplemented with a complete protease inhibitor cocktail (Roche Diagnostics Scandinavia AB, Bromma, Sweden). The lysates were sonicated for 5 min at 4 °C (30 sec ON/30 sec OFF), cleared by centrifugation, and protein concentration was measured by Bradford assay (Sigma-Aldrich AB). Equal amounts of protein (40 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane using a Bio-Rad wet transfer unit (Bio-Rad Laboratories Inc., Sundbyberg, Sweden). Filters blocked with 5% bovine serum albumin (BSA; Saveen Werner, Limhamn, Sweden) were incubated with primary antibodies and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Thermofisher Scientific) as listed in Table S7 , followed by enhanced chemiluminescence assays using the Millipore kit (Merck/Millipore). The original, uncropped immunoblots are listed as the last Supplementary figure.
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!