The protein expression of Sirt1, acetyl-p53, BDNF, and TrkB in the whole retina were analyzed by western blot. After mice eyeballs were enucleated, the retina and sclera were isolated. Protein lysates were extracted using RIPA Lysis Buffer (Thermo Fisher Scientific) supplemented with protease inhibitor (Sigma-Aldrich), according to the manufacturers’ protocol. Briefly, all lysates were quantified, and equal volumes were loaded on NuPAGE 4–12% Bis-Tris gel (Invitrogen) before being transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were blocked with 3% skim milk and incubated with anti-Sirt1 primary antibodies (1:1000; Cell Signaling Technology), anti-acetyl-p53 (1:400; Abcam), rabbit anti-BDNF antibody (1:500; GeneTex, San Antonio, TX, USA), and rabbit anti-TrkB antibody (1:1000; Abcam) overnight at 4°C. Bound proteins were detected using peroxidase-conjugated ECL anti-mouse IgG and anti-rabbit (GE Healthcare UK). Proteins were visualized using a chemiluminescence substrate (Bio-Rad Laboratories) and imaging system (DNR Bio imaging systems, Neve Yamin, Israel). Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The expression ratios of protein bands were quantified using ImageJ, as previous described.68
Imaging system
Manufactured by DNR Bio-Imaging Systems
Sourced in Israel
The DNR Bio-Imaging System is a versatile imaging platform designed for a range of applications in biological and life science research. The system provides high-quality image capture and analysis capabilities, enabling researchers to visualize and study various biological samples. The core function of the imaging system is to capture and process digital images of samples under controlled conditions, allowing for detailed analysis and documentation of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Anti acetyl p53, by Abcam (1 mentions)
Chemiluminescence substrate, by Bio-Rad (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Sds polyacrylamide gel electrophoresis, by Solarbio (1 mentions)
Elc enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit secondary antibody, by Abcam (1 mentions)
Ice cold lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
4 protocols using imaging system
1
Western Blot Analysis of Retinal Proteins
2
Western Blot Analysis of VEGF in Transfected HRMECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected HRMECs were harvested by trypsin digestion and lysed using ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology). Lysate protein concentrations were estimated using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Equal amounts of denatured proteins (20 µg) were resolved using 10% SDS-polyacrylamide gel electrophoresis (Beijing Solarbio Science & Technology Co., Ltd.) and protein bands were transferred to a polyvinylidene fluoride membrane (Beijing Solarbio Science & Technology Co., Ltd.). The membrane was blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at 22±3˚C. Subsequently, the membranes were incubated with an anti-VEGF antibody (1:1,000) overnight at 4˚C, rinsed, and incubated with the horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit; 1:10,000; cat. no. ab205718; Abcam) for 2 h at 22±3˚C. Protein bands were visualized by the addition of ELC enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) in conjunction with an imaging system (DNR Bio-imaging systems, Ltd.). An anti-GAPDH antibody (1:10,000) was used as a loading control.
3
Western Blot Analysis of PTEN
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology) and the protein concentration was determined using BCA protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Denatured proteins (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide 10% gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore). The membrane was blocked with 5% BSA Blocking Buffer (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) and incubated with PTEN primary antibodies (1:1,000; cat. no. ab267787; Abcam) at 4˚C overnight Next, the membranes were incubated with secondary antibody Goat Anti-Rabbit (1:10,000; cat. no. ab205718; Abcam) for 2 h at 25˚C. The bound proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) and detected using an imaging system (DNR Bio-imaging systems Ltd., Ma'ale Hahamisha). β-actin was used as a loading control. Densitometric analysis was performed using ImageJ software (version 1.8.0; National Institutes of Health).
4
Apoptosis Signaling Pathway Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PC9 or PC9/GR cells were lysed using ice-cold RIPA lysis buffer. Lysate protein concentrations were determined using a BCA protein assay kit. Equal amounts of denatured proteins (20 µg) were resolved via 10% SDS-polyacrylamide gel electrophoresis and separated proteins were subsequently transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 1 h at 20°C, incubated with primary antibodies (anti-STAT3, 1:1,000; anti-p-STAT3, 1:1,000; anti-caspase-3, 1:5,000; anti-caspase-3, 1:5000; anti-cleaved caspase-3, 1:500; anti- caspase-9, 1:2,000 and anti-cleaved caspase-9, 1 µg/ml) overnight at 4°C, rinsed with TBS containing 0.05% Tween-20 buffer (Beijing Solarbio Science & Technology Co., Ltd.) twice for 10 min each time, and subsequently incubated with the horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit; 1:10,000) for 2 h at 23±2°C. Protein bands were visualized by the addition of ECL reagent in conjunction with an imaging system (DNR Bio-imaging systems, Ltd.). Anti-GAPDH antibody (1:10,000) was used as a loading control. ImageJ software (version 1.49n; National Institutes of Health) was used for densitometry.
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!