All proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked with 3% bovine serum albumin (BSA) for 2 hours at room temperature. Primary antibodies (Abcam) were diluted with 3% BSA, applied at a 1:1000 dilution to the membranes and incubated at 4°C overnight, the appropriate secondary antibodies (Cell Signaling Technology) were incubated for 1 hour at room temperature. Protein expression was detected using the enhanced chemiluminescent HRP substrate (Cell Signaling Technology) and imaged with a Chemiluminescence Imaging System (Bio-Rad Laboratories).
Appropriate secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Appropriate secondary antibodies are laboratory reagents used to detect and visualize target proteins in biological samples. They are designed to bind to the Fc region of primary antibodies, allowing for the amplification and detection of the primary antibody signal. These secondary antibodies are available in a variety of formats, such as enzyme-conjugated or fluorescently labeled, to enable diverse detection methods in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemiluminescence imaging system, by Bio-Rad (3 mentions)
Protease and phosphatase inhibitor, by Merck Group (3 mentions)
Ripa lysis buffer, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Imagej software, by Media Cybernetics (1 mentions)
Protease inhibitor, by Abcam (1 mentions)
Enhanced chemiluminescence ecl kit, by Merck Group (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Primary antibodies against runx2, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Elpis Biotech (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
22 protocols using appropriate secondary antibody
1
Western Blot Protocol for Protein Analysis
2
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were collected and prepared as described previously [45 (link), 46 (link)]. Briefly, 661W cells were harvested and lysed in a Tris lysis buffer (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, and 1 Na3VO4, pH 7.5. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The primary antibodies used in this study were DRP1 (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), MFN2 (1 : 1000 Abcam, Cambridge, MA, USA), MCU (1 : 1000 Abcam), and actin (loading control; 1 : 1000, Cell Signaling Technology). Blots were visualized using appropriate secondary antibodies (Cell Signaling Technology) at 1 : 1000 conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). Band intensities were quantified by densitometry using Scion Image (NIH, Bethesda, MD, USA).
3
Western Blot Analysis of NDUFS1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissue samples were lysed in ice‐cold RIPA lysis buffer containing protease inhibitor. An equal amount of total protein from each sample (20 µg) was loaded on 10% SDS‐PAGE gels and then transferred to PVDF membranes. The membranes were blocked with 5% BSA for 2 h at room temperature and then incubated with commercially available primary antibodies for detection of NDUFS1 (Cell Signaling Technology, USA), followed by the appropriate secondary antibodies (Cell Signaling Technology, USA). GAPDH was taken as internal control and protein bands were analyzed by Image J software (NIH, USA).
4
Protein Expression Analysis in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The eWAT and liver tissues were homogenized in RIPA lysis buffer (Millipore, Billerica, MA, USA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). The total protein concentration was determined using a BCA Protein Assay Kit (Pierce, Bonn, Germany). The lysates were blotted overnight at 4°C with primary antibodies (Supplementary Table 2), and then incubated with appropriate secondary antibodies (Cell Signalling Technology, Danvers, MA, USA). The proteins were visualized by chemiluminescence (Millipore) and imaged using a gel imaging system (ChemiDoc™ XRS; Bio-Rad, Hercules, CA, USA). Pixel intensities of the immunoreactive bands were quantified using Quantity One software (ver. 4.5.2; Bio-Rad).
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were homogenized in RIPA lysis buffer (Millipore, Billerica, Massachusetts, USA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). The lysates were blotted with primary antibodies overnight at 4°C (online supplementary table 3 ), and then incubated with appropriate secondary antibodies (Cell Signaling Technology, Danvers, Massachusetts, USA). Proteins were visualized via chemiluminescence (Millipore) and imaged using a CCD camera system (GE Healthcare Japan, Tokyo, Japan). Pixel intensities of immunoreactive bands were quantified using ImageQuant TL software (ImageQuant LAS 4000; GE Healthcare).
6
Western Blot Analysis of MCM5 and Actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extraction was performed using a radioimmunoprecipitation assay (RIPA) buffer obtained from Beyotime Biotechnology (Shanghai, China). After quantification of the extracted proteins, 30 μg of total protein was separated using 10–12% SDS-PAGE gels and subsequently electrophoretically transferred onto either a 0.2μm or 0.45μm PVDF membrane sourced from Millipore Sigma (Burlington, MA, USA). The membranes were then blocked with 5% fat-free milk for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies acquired from Abcam (Cambridge, MA, USA): MCM5 (ab75975, 1:500) and Actin (ab115777, 1:2000). Appropriate secondary antibodies (Cell Signaling Technology, Dallas, TX, USA) were incubated with the membranes for 2 hours at room temperature. Subsequently, the protein bands were visualized using chemiluminescence and quantified using ImageJ software from Media Cybernetics (Silver Springs, MD, USA).
7
Protein Expression Analysis of BMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular proteins were extracted, and western blotting was performed as previously reported (Mao et al., 2017 (link)). Briefly, total proteins were isolated from BMSCs with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Beijing, China) containing protease inhibitors (Abcam, Cambridge, United Kingdom). Membranes were incubated at 4°C overnight with primary antibodies against RUNX2 (1:2,000; Cell Signaling Technology, Danvers, MA, United States), OCN, OPN, and SORBS1 (1:1,000; Proteintech, Wuhan, China). GAPDH (1:3,000; Cell Signaling Technology, Danvers, MA, United States) was used as the internal control. Appropriate secondary antibodies (1:3,000; Cell Signaling Technology, Danvers, MA, United States) were incubated with the blots at 20–25°C for 1 h. After rinsing with Tris-buffered saline (TBS) containing 0.05% (w/v) Tween-20 (TBST), the signals were detected using an enhanced chemiluminescence (ECL) kit (Millipore, Darmstadt, Germany) and a chemiluminescence system (Bio-Rad Laboratories, Hercules, CA, United States) and analyzed with Image Lab v6.0 software.1
8
Analyzing NF-κB Activation and IL-6 Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA buffer (ELPIS Biotech) containing protease and phosphatase inhibitor cocktails (Roche Diagnostics, Mannheim, Germany) and equal amount of protein mixed with sodium dodecyl sulphate sample buffer were boiled for 5 minutes. Samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane. The membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, and then incubated overnight with following primary antibodies at 4°C: phospho-nuclear factor (NF)-κB p65 (Ser536) from Cell Signaling Technology (Danvers, MA, USA) and beta-actin from SantaCruz Biotechnology (Dallas, TX, USA). Membranes were washed using TBS-T and further incubated with appropriate secondary antibodies (Cell Signaling Technology) for 1 hour at room temperature. The immune-reactive proteins were detected using a chemiluminescence kit. ELISA to detect the levels of interleukin (IL)-6 in cell supernatant was performed according to the manufacturer's protocol (R&D Systems; cat. No. DY206-05).
9
NF-κB Activation in Macrophages
10
Western Blot Analysis of WASH Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in ice‐cold lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) plus protease inhibitor cocktail (Sigma‐Aldrich, St Louis, MO, USA). An equal amount of total protein from each sample (50 μg) was resolved on 10% SDS‐PAGE gel and then transferred to nitrocellulose membrane. The membrane was blocked with 5% non‐fat milk and then incubated with commercially available primary antibodies for the detection of WASH (Abcam, Cambridge, UK) and β‐actin (Cell Signaling Technology, Beverly, MA, USA), followed by the appropriate secondary antibodies (Cell Signaling Technology). Protein expression was detected using enhanced chemiluminescent HRP substrate (Thermo Fisher Scientific) and photographed with a Chemiluminescence Imaging System (Bio‐Rad Laboratories, Hercules, CA, USA).
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!