Total proteins were extracted from cells in RIPA buffer with protease inhibitor and then quantified using a BCA protein assay kit (Pierce, Santa Cruz, CA, USA). SDS-PAGE was used to separate equivalent amounts of protein and then deposited onto PVDF membranes (Millipore, Billerica, USA). After blocking with 5% nonfat milk, the primary antibodies were incubated with the PVDF membrane overnight at 4°C, followed by 1 h at 25°C with the appropriate secondary antibody. Enhanced chemiluminescence was used to see protein bands (Millipore, Billerica, MA, USA). Proteintech provided the primary antibodies against Cyclin D1, CDK2, P21, and β-actin (Chicago, IL, USA). Abcam provided antibodies against PCNA, Cox-2, MMP-2, and MMP-9 (Cambridge, MA, USA). Novus Biologicals provided the antibody against MSL2 (Littleton, CO, USA). β-Actin was used as the internal reference. Protein bands were visualized using Enhanced chemiluminescence (MilliporeSigma) and quantified using ImageJ software (version 4.3; National Institutes of Health).
Enhanced chemiluminescence
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, Ireland, Taiwan, Province of China, Japan
Enhanced chemiluminescence is a laboratory technique that utilizes light-emitting chemical reactions to detect and quantify specific biomolecules, such as proteins and nucleic acids. It provides a sensitive and reliable method for analysis and visualization of target analytes in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (89 mentions)
Polyvinylidene difluoride membrane, by Merck Group (32 mentions)
Polyvinylidene fluoride membrane, by Merck Group (27 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (27 mentions)
Nitrocellulose membrane, by Merck Group (25 mentions)
Ripa lysis buffer, by Beyotime (24 mentions)
Bca protein assay kit, by Beyotime (23 mentions)
Protease inhibitor cocktail, by Roche (22 mentions)
Ripa buffer, by Beyotime (19 mentions)
Bca kit, by Thermo Fisher Scientific (17 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (17 mentions)
Protease inhibitor cocktail, by Merck Group (14 mentions)
Gapdh, by Cell Signaling Technology (14 mentions)
β actin, by Cell Signaling Technology (13 mentions)
Image lab software, by Bio-Rad (13 mentions)
Ripa buffer, by Thermo Fisher Scientific (12 mentions)
β actin, by Abcam (11 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (10 mentions)
Ripa buffer, by Solarbio (9 mentions)
Protease inhibitor, by Roche (8 mentions)
548 protocols using enhanced chemiluminescence
1
Protein Expression Analysis by Western Blot
2
Protein Expression Analysis via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer, which contained a protease inhibitor (Cell Signaling Technology, Danvers, MA, USA), was applied for cell lysis. The protein concentrations were determined using a BCA protein assay kit (KeyGEN BioTECH, Nanjing, China). SDS-PAGE (8–10%) was used to separate the proteins, which were then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). The primary antibodies included HIF-2α (1:500, ab109616, Abcam, Cambridge, UK), c-Myc (1:500, #13987, Cell Signaling Technology), GOT1 (1:1000, #14886-1-AP, Proteintech, Rosemont, IL, USA), and β-actin (1:5000, #AP0060, Bioworld, Bloomington, MN, USA). Enhanced chemiluminescence (Merck Millipore) was used to evaluate the signal, and analysis was performed using the ImagePro Plus software (Media Cybernetics, Rockville, MD, USA). Each target gene protein was semi-quantitatively estimated, compared with β-actin as an internal loading control.
3
Hippocampal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hippocampus preparation and Western blotting was conducted as described previously by our previous work [13 (link)]. Briefly, the tissue was dissected and homogenized in ice-cold radioimmunoprecipitation assay buffer. Homogenates were spun at 14,000×g for 15 min at 4 °C, and the supernatants were collected. Later, 20 μg/lane of sample was run on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to microporous polyvinylidene fluoride membranes (0.45 μm, F. Hoffmann-La Roche Ltd., Germany). Immunoblots were probed with the specific antibodies as shown in the section “Primary antibodies ,” and then incubated with peroxidase-conjugated species-specific anti-IgG secondary antibodies. After visualizing by enhanced chemiluminescence (EMD Millipore Co., USA), the intensity values of the immunoreactive signals were quantified by using Gel-Pro 3.1 software (Media Cybernetics Inc., USA).
4
Testosterone Signaling Modulation by Conditioned Media
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To test direct impact of CM on testosterone signaling, LNCaP cells were treated with TP (100 ng/mL) and dihydrotestosterone (10 ng/mL) with or without CM (100 µg/mL) for 24 h. Cells were lysed using RIPA buffer (Cell Signaling Technology, Tokyo, Japan), and the constituent proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA). After blocking with Bullet Blocking One (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse polyclonal antiphosphorylated androgen receptor (p-AR) antibody (1:500, Merck Millipore), or mouse monoclonal anti-GAPDH (1:5000, Fujifilm Wako Pure Chemical Corp.). Immunoreactive bands were detected using enhanced chemiluminescence (Merck Millipore) after incubation with horseradish peroxidase-labeled goat antimouse IgG (1:5000, Vector Laboratories, Burlingame, CA, USA).
5
Immunoblot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment, cells were washed two times in cold phosphate-buffered saline and collected on ice. Cells were lysed and immunobloted as described previously.36 (link) Equal amounts of total protein (30 μg) was separated by 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis minigel electrophoresis and transferred onto nitrocellulose membranes. Membranes were sequentially probed with indicated primary antibodies and corresponding mouse or rabbit horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich). Blots were then developed using enhanced chemiluminescence (Merck Millipore, Billerica, MA, USA).
6
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Subsequent to the indicated treatments, protein of cells was harvested with RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime) and determined using a bicinchoninic acid assay kit (Beyotime). Proteins were separated with 10% or 12% SDS-PAGE gels and transferred PVDF membranes (Merck Millipore, Billerica, MA, USA), which were blocked with 5% non-fat milk for 1 h at room temperature. The membranes were incubated with primary antibody at 4°C overnight and then blotted with secondary antibody for 2 h at room temperature. The bands were detected using enhanced chemiluminescence (Merck Millipore). The primary antibodies against p62 (1:1000), LC3 (1:1000), and β-actin (1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).
7
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissue samples were lysed in ice-cold radioimmunoprecipitation assay buffer supplemented with the Protease Inhibitor Cocktail (Sigma-Aldrich). The lysates were resolved in sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk and incubated with anti-PAX8, anti-E-cadherin, anti-vimentin, anti-vascular endothelial growth factor (VEGF), anti-FOXM1, anti-FOXC2, anti-FOXF1, anti-FOXL1, and anti-β-actin (Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Signals were developed by enhanced chemiluminescence (Merck Millipore, Darmstadt, Germany).
8
Western Blotting of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from PASMCs. Briefly, the PASMCs were washed with 1X PBS once and each well in the 12-well cell culture plate was lysed with 100 µl of ice cold radioimmunoprecipitation assay buffer (25 mM Tris-Cl, pH 7.4, 150 mM NaCl, 50 mM KCl, 1% SDS, 2 mM EDTA, 0.5% glycerol, 50 mM NaF) with 1:100 (vol/vol) dilution of the proteinase inhibitor and phosphotase I and II inhibitor mixture (Sigma-Aldrich; Merck KGaA). This was then briefly vortexed and placed on ice for 15 min, spun down at 8,000 × g for 15 min to pellet the debris and the supernatant was collected for the western blotting. The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's instructions. Then, 30 µg protein lysate was boiled with 500 mM DTT in 2X sample buffer for 5 min, separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline containing 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature for 30 min, followed by incubation with the appropriate primary antibody (1:1,000) overnight at 4°C. β-actin served as an internal control. Following this, membranes were incubated with a secondary antibody (1:5,000) at room temperature for 1 h. Proteins were visualized by Enhanced Chemiluminescence (EMD Millipore, Billerica, MA, USA).
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW264.7 and MH‐S cells were plated in 12‐well plate. Collected the cell pellet and extracted proteins with RIPA buffer. The cell lysates were centrifuged at 14 000 × g for 20 minutes at 4°C. The total protein concentration was determined by BCA assay kit (KeyGen Biotech). Equal amounts of 20 µg protein lysate were added into the wells of the SDS‐PAGE gels. The separated cells were then transferred onto a PVDF membrane and blocked with 5% blotting milk in PBS buffer for 1 hour, and incubated with the diluted primary antibody in 0.5% PBST buffer overnight at 4°C, then added the secondary antibody (1:1000) for 2 hours at room temperature. The intensity of the band signals were detected by enhanced chemiluminescence (Merck Millipore) and exposed with ChemiDoc™ MP Imager (Bio‐Rad).
10
Protein Immunoblotting and Densitometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was prepared as described before (Hagenbuchner et al., 2016a (link)). Proteins were separated by SDS-PAGE and blotted on nitrocellulose membrane (GE Healthcare, Chalfot, UK). After blocking, membranes were incubated with primary antibodies directed against Bim, Noxa, DEPP1, SESN3, FOXO3, GAPDH or αTubulin (Supplementary file 2 ), washed and incubated with horseradish-peroxidase conjugated secondary antibody (GE Healthcare, UK). The immunoblots were developed by enhanced chemiluminescence (Merck, Vienna, Austria) according to manufacturer’s instructions and analyzed using an AutoChemi detection system (UVP, Cambridge, UK). Densitometry was performed using Labworks software version 4.5 (UVP, UK).
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!