A total of 20 μg protein samples, obtained from the ischemic brain tissues, were loaded onto a 12% SDS-PAGE gel. After the electrophoresis, proteins were transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked with 5% nonfat milk in TBST for 1 h and then incubated with primary antibodies against BDNF, Akt, p-Akt, Bcl-2, Bax, caspase-3, and β-actin (1 : 1000, Bioworld, USA) overnight at 4°C. Subsequently, blots were incubated with HRP-conjugated secondary antibody at room temperature for 1 h. The protein bands were visualized with ECL chemiluminescence system (Thermo Scientific, Rockford, IL). The optical density was quantified by performing the National Institute of Health ImageJ software. β-actin was performed as a loading control.
Ecl chemiluminescence system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL chemiluminescence system is a laboratory equipment used to detect and analyze specific proteins or molecules in a sample. It utilizes a chemical reaction that produces light, which is then measured and quantified. The system is commonly used in various applications, such as Western blotting, immunoassays, and protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx stain free gel, by Bio-Rad (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Polyvinylidene fluoride membrane, by Roche (3 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protein extraction reagent, by Merck Group (2 mentions)
Rabbit anti actin antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti gadph antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti cxcr4 antibody, by Abcam (2 mentions)
Lysis buffer, by Ozyme (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Roche (2 mentions)
Tc dish, by Sarstedt (2 mentions)
Hrp conjugated secondary antibody, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti mouse igg hrp, by GE Healthcare (1 mentions)
Anti sqstm p62, by Cell Signaling Technology (1 mentions)
39 protocols using ecl chemiluminescence system
1
Quantifying Brain Tissue Protein Profiles
2
Western Blotting for p62 and α-Tubulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as described before [19 (link)]. For detection of the proteins following primary and secondary antibody were used: anti-SQSTM/p62 (#5114, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (T5168, 1:10,000, Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG-HRP (NXA931, 1:10,000, GE Healthcare, Little Chalfont, UK), anti-rabbit IgG-HRP (Cell Signaling Technology, Danvers, MA, USA, #7074, 1:5000). Signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. Relative optical density of the signal intensity of the bands was quantified with Quantity One 4.6 software (Bio-Rad, Hercules, CA, USA) and normalized first, against corresponding signal of α-tubulin (loading control), and second, against the signal of control sample. Mean values of two independent experiments ± SEM are presented. SEM values were calculated using GraphPad Prism software v. 5.01 (GraphPad Prism software Inc., La Jolla, CA, USA).
3
Quantification of Autophagy-Related Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total EPC protein was extracted and quantified by RIPA Lysis Buffer (Beyotime Biotechnology, China) containing protease inhibitors (Roche) and BCA assay kit (I Thermo, USA) separately. Protein extracts were separated by SDS-PAGE, transferred to PVDF membranes (Roche, Indianapolis, IN, USA). The following antibodies were used: anti-Beclin1 antibody (1 : 1000; ImmunoWay, USA), anti-Atg5 antibody (1 : 1000; ImmunoWay, USA), rabbit anti-LC3 antibody (1 : 1000; Cell Signaling Technology, USA), and anti-ACTB antibody (1 : 1000; Cell Signaling Technology, USA). Proteins were visualized with HRP-conjugated anti-rabbit IgG or anti-mouse IgG (1 : 3000; Cell Signaling Technology, USA), followed by the use of the ECL chemiluminescence system (Thermo). And the level of protein was analyzed by using Image J.
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
Total EPCs protein were extracted and quantified by protein extraction reagent (Merck) and bicinchoninic acid protein assay kit (Thermo Fisher) separately. Protein extracts were subjected to SDS‐PAGE, transferred to polyvinylidene fluoride membranes (Roche). The following antibodies were used: rabbit anti‐CXCR4 antibody (1:500; ABCAM, USA), rabbit anti‐actin antibody (1:2000; Cell Signaling Technology), rabbit anti‐VEGFa antibody rabbit (1:500; Santa Cruz, USA), rabbit anti pan protein kinase B (1:1000, Abcam), p‐Akt (1:2000, Ser473; Abcam), rabbit anti human PDGF B (1:2000, Abcam), goat anti human FGF 23 (1:800, Abcam), mouse anti human eNOS (1:500 Abcam), p‐eNOS (1:500 Ser1177; Abcam), and rabbit anti‐GADPH antibody (1:3000; Cell Signaling Technology). Proteins were visualized with HRP‐conjugated anti‐rabbit or anti goat IgG (1:2000; Cell Signaling Technology), followed by use of the ECL chemiluminescence system (Thermo).
5
Protein Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were anesthetized by isoflurane inhalation, and the brains were immediately recovered after decapitation and quickly placed in dry ice. Tissue and cell samples were homogenized in a lysis buffer (Ozyme, Saint-Quentin-en-Yvelines, France) or RIPA buffer (Sigma-Aldrich) and analyzed by electrophoresis and protein transfer onto a PVDF membrane (Thermo Fisher Scientific). For all experiments, 30 μg of proteins was used. Membranes were probed with polyclonal antibodies against TH (Millipore) diluted 1:200, anti-active caspase 3 diluted 1:200 (Cell Signaling, Danvers, USA), phosphoTH-Ser31 diluted 1:500, phosphoTH-Ser40 (Ozyme) diluted 1:400, BiP/GRP78 (Sigma-Aldrich) diluted 1:100, DJ-1/PARK7 (Millipore) diluted 1:100, α-synuclein (Sigma-Aldrich) diluted 1:250, anti-H3K27me diluted 1:500 (Diagenode, Ougrée, Belgium) and anti-H3 diluted 1:500 (Abcam). After incubation with Alexia conjugated secondary antibodies (Thermo Fisher Scientific) diluted at 1:1000, the resulting immune complexes were visualized using the ECL chemiluminescence system (Thermo Fischer Scientific). An antibody against α-tubulin (Sigma-Aldrich) or GAPDH (Sigma-Aldrich) diluted at 1:1000, was used as a control to ensure equal protein loading.
6
CXCR7 Pathway Regulation in Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total EPC protein was extracted and quantified using RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitors and BCA assay kit (Thermo Fisher Scientific, USA) separately. Protein extracts were separated via SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Indianapolis, IN, USA). The following antibodies were used: anti-CXCR7 antibody (1 : 1000; Affinity, USA), anti-p38 mitogen-activated protein kinase (MAPK) antibody (1 : 1000; Affinity, USA), anti-p-p38 MAPK antibody (1 : 1000; Affinity, USA), anti-cleaved casepase-3 antibody (1 : 1000; Cell Signaling Technology, USA), anti-GAPDH antibody (1 : 1000; Cell Signaling Technology, USA), and HRP-conjugated anti-rabbit IgG (1 : 3000; Cell Signaling Technology, USA). Protein bands were visualized using an ECL chemiluminescence system (Thermo Fisher Scientific, USA).
7
Protein Isolation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated in RIPA buffer. Extracted protein was separated on a 10% SDS-PAGE gel, and size-fractioned protein samples were transferred to a nitrocellulose membrane. Membranes were blocked with 5% skim milk (Sigma-Aldrich) and incubated with primary antibodies overnight at 4 °C. After reaction with the appropriate horseradish peroxidase-conjugated secondary antibodies, signaling was visualized using an ECL chemiluminescence system (Thermo Fisher Scientific).
8
Protein Separation and Detection in 22Rv1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The assay was performed as previously reported [59 (link)]. 22Rv1 cells were seeded in Petri dishes (ø 6 cm, 106 cells/well in 5 mL/dish), incubated overnight and treated with the compounds in fresh culture media (5 mL/dish) for indicated time. Then, cells were harvested and the proteins were extracted and separated using PAGE in gradient ready-made gels. The proteins were then transferred onto ø 0.2 µm pore PVDF membrane, followed by blocking and treated with primary and secondary antibodies. The signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA). The antibodies used are listed in Table 2 .
9
Cell Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preparation of protein extracts and Western blotting were performed as described previously with slight modifications [44 (link)]. In brief, 1 × 106 cells/well were seeded in Petri dishes (ø 6 cm, 5 mL/dish). Cells were harvested, proteins were extracted, subjected to electrophoresis in 10-15% SDS-polyacrylamide gels at 120 V, and transferred from gel to a 0.2 μm pore PVDF membrane. The membrane was blocked and incubated with the primary antibody according to the manufacturer's protocol (for antibodies used, see the supplementary). After washing, the membranes were incubated with the corresponding secondary antibody for 1 h at RT. Signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol.
10
Molecular Profiling of Vascular Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein expression of NF-κB, PPAR-γ, CD36, MMP-9, ICAM-1, VCAM-1, p38, and P-p38 were detected by Western blot. Experimental procedures were strictly in accordance with the BCA kit (Solarbio, Beijing, PC0020) manual. Carotid artery was sonicated with pre-cooled lysate. The supernatant was centrifuged (13 000 rpm, 30 min) at 4°C, and the protein concentration was detected by BCA method. The total proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Merck, Darmstadt, Germany). The PVDF membrane was immersed in 5% milk powder in TBST buffer (20 mM Tris, 137 mM NaCl, pH 7.6, with 0.1% Tween 20) and blocked for 1 h. Then, the PVDF membrane was incubated with the corresponding primary antibody (1: 1000, Abcam) for 2 h at room temperature followed by washing 3 times with TBST buffer for 10 min at room temperature, and the corresponding HRP-conjugated secondary antibody (1: 2000, Abcam) was added and incubated for 1 h at room temperature. The ECL chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the signal on the specimen membrane and the X-ray film recorded the experimental results. The spectral densities of the bands were analyzed using AlphaView SA software (Thermo Fisher Scientific, Waltham, MA, 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!