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).
Protocol detail
Quantitative Western Blot Analysis
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
polyvinylidene difluoride membranes
RPE65
TIMP3
horseradish peroxidase conjugated
Azure C500
LiCor Odyssey 3.0
Image Studio Lite version 5.2
After image
Antibodies
Azure
Cell
Gels
Horseradish peroxidase
Lam 1
Membranes
Polyvinylidene difluoride
Sds page
Timp3
Western blot
Corresponding Organization : University of Rochester
Other organizations : Queensland University of Technology, Queensland Eye Institute, University of Wisconsin–Madison, Smith-Kettlewell Eye Research Institute
Top 5 similar protocols
Protocol cited in 1 other protocol
Variable analysis
independent variables
- Total cell lysate and ECM were isolated
- Resolved gels were transferred to polyvinylidene difluoride membranes and probed with the following primary antibodies: ACTN, BEST1, COL4, CRALBP, EZR, LAM, OCLN, RPE65, RHO, and TIMP3
- SDS-PAGE in 4% to 20% gels as previously described.17 (link)
- 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.
- 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).
- CRALBP (1:10000)25 (link)
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!