Samples were loaded onto SDS-polyacrylamide gels (NuPage 4%–12% Bis-Tris Protein Gels, Novex) and transferred to PVDF membranes (Immobilon-FL), which were incubated in primary antibody (1:500 mouse anti-RhoA, ARH04, Cytoskeletal Inc.; 1:1000 mouse anti-β-tubulin, Developmental Studies Hybridoma Bank; 1:1000 mouse anti-Rac1 BD Bioscience; or 1:1000 rabbit anti-RhoC, D40E4, Cell Signaling) overnight. The blots were subsequently incubated with secondary antibody (1:10000 goat-anti-mouse; Rockland, 610-731-124; 1:10000 goat anti-mouse Jackson Labs, 715-036-151; or 1:20000 goat anti-rabbit LiCOR, 925-68071) and signal was assessed using either an Odyssey (LI-COR) or a ChemiDocX (BioRad) system.
Chemidocx
Manufactured by Bio-Rad
Sourced in United States
The ChemiDocX is a compact and versatile imaging system designed for capturing and analyzing a variety of sample types, including chemiluminescent, fluorescent, and colorimetric gels and blots. The system features an advanced CCD camera, precision optics, and powerful image analysis software to provide high-quality images and accurate quantification.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Mouse anti rac1, by BD (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Mouse anti β tubulin, by Developmental Studies Hybridoma Bank (1 mentions)
Ripa lysate buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Solarbio (1 mentions)
Hikari solution, by Nacalai Tesque (1 mentions)
3 protocols using chemidocx
1
Western Blot Analysis of Rho GTPases
2
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins were obtained with RIPA lysate buffer (Beyotime) including a protease inhibitor, and the concentration was analyzed through the BCA method. 40 μg proteins was denatured by heating at 100°C for 3 min and then was separated on 10% SDS-PAGE (Solarbio) through electrophoresis. After transfer to the PVDF membrane (Millipore), the proteins were blocked with 5% skimmed milk and addressed with primary antibodies overnight at 4°C and HRP-secondary antibody (Cell Signaling Technology) for 2 h. After treatment with an ECL kit (Thermo Scientific), the results were confirmed through ChemiDoc-X (Bio-Rad).
3
Ras and Caspase-3 Expression in NPC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After infection with MOI 0.1 and 10 of PRV7S for 6, 24 and 48 h, respectively, NPC (CNE1 and TWO1) and NCNP (NP69) cells were collected for Western blot analysis. The cells were lysed by using ice-cold RIPA buffer with protease and phosphatase inhibitors. The total protein concentrations were measured with the Bradford method. Protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 12% polyacrylamide gels. Next, the gels were transferred to PVDF membrane via wet transfer. After wet transfer, the membrane was washed with TBST and 5% bovine serum albumin to prevent from unspecific bindings. Furthermore, the membranes were probed with primary antibodies (1:1000 dilution) in Hikari solution (Nacalai Tesque, Japan). The primary antibodies against N/H/K-Ras (Elabscience, USA) and Caspase-3 (FineTest, China) were used, while β-actin (FineTest, China) was used as housekeeping protein. After primary antibodies incubation, the membrane was washed 3 times and incubated with HRP-linked secondary antibody. Lastly, Western blot bands were imaged and visualized by using ChemiDocX (Bio-Rad Laboratories, Inc. 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!