Sciatic nerve segments were lysed and homogenised in Cell Lytic MT lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). Protein samples (20 μg) were treated with reducing agent and sample buffer (Invitrogen) before 10 min incubation at 90 °C. Once cooled samples were run on SDS-PAGE gels (Invitrogen, NuPAGE 4–15% Bis-Tris gel) and transferred to nitrocellulose using an iblot (Invitrogen). The Western blot was performed using standard techniques, visualised and captured using the Azure C500 (Azure Biosystems) and densitometry analysis was performed using Fiji. Whole protein was used as a loading control with the amido black stain (Sigma) where densitometry analysis for each entire lane was used to normalise in Fiji, after being captured using the AzureC500 (Azure Biosystems)67 (link).
Azure c500
Manufactured by Azure Biosystems
Sourced in United States
The Azure C500 is a compact, easy-to-use imaging system designed for a wide range of applications in life science research. It features a high-resolution camera, a variety of imaging modes, and intuitive software to capture and analyze images of gels, membranes, and other samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (15 mentions)
Tris glycine gel, by Thermo Fisher Scientific (10 mentions)
Hmgb1, by Abcam (8 mentions)
Chemiluminescence reagent kit, by Thermo Fisher Scientific (7 mentions)
Chemilluminescence reagent kit, by Thermo Fisher Scientific (7 mentions)
Epac1, by Abcam (6 mentions)
Il 1β, by Abcam (5 mentions)
Occludin, by Abcam (4 mentions)
Cleaved caspase 1, by Abcam (4 mentions)
Nlrp3, by Abcam (4 mentions)
Pre cast tris glycine gel, by Thermo Fisher Scientific (3 mentions)
Tnf α, by Abcam (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Igfbp3, by Abcam (2 mentions)
Protease and phosphatase inhibitor cocktail, by Cell Signaling Technology (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (2 mentions)
Renaturing buffer, by Thermo Fisher Scientific (2 mentions)
Developing buffer, by Thermo Fisher Scientific (2 mentions)
Ab109415, by Abcam (1 mentions)
38 protocols using azure c500
1
Western Blot Analysis of Sciatic Nerve Proteins
2
Epac and NF-κB Regulation in Retina
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole retinal lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated on a precast Tris-glycine gel (Invitrogen) and blotted on nitrocellulose membrane. After blocking in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and 5% (w/v) bovine serum albumin, the membranes were treated with Epac1 (ab109415), Epac 2 (ab193665, Abcam), total nuclear factor kappa beta (NFκB; #4764), phosphorylated NFκB (Ser 536, #3303), phosphorylated IκB (Ser32, #2859), total IκB (#4812, Cell Signaling, Danvers, MA), and beta actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected with a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA), and data were acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software.
3
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole retinal lysates from mice or cell culture lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated onto a precast Tris-glycine gel (Invitrogen) and blotted on nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, and 0.1% Tween-20) and 5% (w/v) bovine serum albumin (BSA), the membranes were treated with acetyl-lysine (1:500, Cat #80178) and IGFBP-3 (1:500, Cat# 76,001; Abcam) or GAPDH and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies overnight followed by incubation with secondary antibodies (anti-rabbit-horseradish peroxidase; Promega, Madison, WI) for 0.5–1.0 h. Antigen-antibody complexes were detected with a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA), and data were acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software. A representative blot for each treatment group is shown.
4
Western Blot Analysis of Retinal Proteins
5
Zymography Assay for Protease Activity
6
Western Blot Analysis of Retinal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After appropriate treatments and rinsing with cold PBS, REC or whole retinal lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein from the cell extracts were separated on the precast tris-glycine gel (Invitrogen), blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (wt/vol) BSA, the membranes was treated with Epac1, PKCzeta, Occludin, ZO-1, VEGF (all from Abcam, San Francisco, CA, USA), or beta actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA, USA). Data was acquired using an Azure C500 (Azure Biosystems, Dublin, CA, USA). Western blot analyses were done using Image Studio Light software (LI-COR, Lincoln, NE, USA).
7
Apoptotic Pathway Analysis of TXA Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multi-apoptotic protein detection arrays (Human Apoptosis Antibody Array Kit; R&D Systems, Inc.) were used to determine possible apoptotic pathways involved in cell death according to the manufacturer’s instructions. Cells were treated with 0 mg/ml, 1 mg/ml, 50 mg/ml, or 100 mg/ml TXA for one hour. The protein content of each well was quantified using a Micro bicinchoninic acid assay (BCA) Protein Assay Kit (Thermo Fisher Scientific) and read on a microplate reader at 540 nm. Lysate protein concentrations were then normalized in lysis buffer. Cells were cultured in 12-well plates for one hour, with increasing concentrations of TXA with medium (0 mg/ml, 1 mg/ml, 50 mg/ml, or 100 mg/ml). Cell supernatants were aspirated and discarded. After blocking of non-specific binding at room temperature for one hour, antibody array membranes were incubated with the cell lysate (1.5 ml) at 4°C overnight and then with a diluted solution of horseradish peroxidase (HRP)-conjugated streptavidin at room temperature for 30 minutes. Visualization of protein expression was carried out by chemiluminescence and signal intensity was quantified using Luminex (Azure c500; Azure Biosystems, Dublin, California). The immunoreactive band density was analyzed using WinImage studio version 5.2 (LI-COR Biosciences, Lincoln, Nebraska).
8
Quantitative Immunoblotting of Retinal Proteins
9
Western Blot Analysis of Insulin Signaling
10
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysate and ECM were isolated and resolved by SDS-PAGE in 4% to 20% gels as previously described.17 (link) Resolved gels were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA) and probed with the following primary antibodies: ACTN (1:750; Santa Cruz Biotechnology, Dallas, TX, USA), BEST1 (1:500; Millipore, Billerica, MA, USA), COL4 (1:1000; Abcam, Cambridge, MA, USA), CRALBP (1:10000),25 (link) EZR (1:1000, Cell Signaling Technology, Danvers, MA, USA), LAM (1:1000; Abcam), OCLN (1:1000; Thermo Fisher Scientific), RPE65 (1:500; Millipore), RHO (1:500; Millipore), and TIMP3 (1:250; Abcam). Secondary antibodies were host-specific near-infrared (1:12,500) (LiCor, Lincoln, NE, USA) or horseradish peroxidase conjugated (1:10,000) (Jackson ImmunoResearch, West Grove, PA, USA) and signals were detected on the LiCor Odyssey or the Azure C500 (Azure Biosystems, Dublin, CA, USA) imaging systems. After image acquisition, Western blot data were analyzed quantitatively using LiCor Odyssey 3.0 and/or Image Studio Lite version 5.2 (LiCor) and Microsoft Excel (Redmond, WA, 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!