Intracellular staining was performed on fixed and permeabilized RMS and RT cell suspensions with Transcription Factor Buffer Set (BD Biosciences, Jose, CA) prior to staining with fluorochrome-conjugated anti-human antibodies following manufacturer's protocol with minor modifications. Briefly, cells were incubated in Fix/Perm buffer for 40 min, washed with Perm/Wash buffer, and incubated for 15 min with 2% rabbit/2% mouse normal sera to block nonspecific binding. Cells were then stained for 2 hrs with mixture of fluorochrome-conjugated antibodies in Perm/Wash buffer: Bcl-2 (clone 100) from BioLegend, MYOD1 (polyclonal) and NOG (polyclonal) from Bioss Antibodies, MYOD1 (clone SPM427) and myogenin (clone MGN185 + F5D) from Novus Biologicals, and ID1 (clone B-8) from Santa Cruz Biotechnology, followed by staining with PE-streptavidin (Invitrogen) if biotinylated antibody was included in the staining mixture. The optimal antibody dilution was established by titration curve. Matching isotype controls were prepared as per standard flow cytometry protocols to determine background signals. All steps were performed on ice and samples kept in the dark. Flow cytometry was performed using an 8-color Stratedigm (San Jose, CA) S1000EX apparatus. Data were analyzed using CellCapTure software (Stratedigm).
Bcl 2 clone 100
Manufactured by BioLegend
Sourced in United States
Bcl-2 (clone 100) is a lab equipment product offered by BioLegend. It is a monoclonal antibody that binds to the Bcl-2 protein, which is involved in the regulation of apoptosis (programmed cell death). The core function of this product is to detect and quantify Bcl-2 expression in various cell types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Cellcapture software, by Stratedigm (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Sox2 clone d6d9, by Cell Signaling Technology (1 mentions)
P akt clone d9e, by Cell Signaling Technology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
2 protocols using bcl 2 clone 100
1
Intracellular Protein Expression Profiling
2
Quantitative Protein Analysis in SNU478 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from SNU478 cells was extracted by PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea), and protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich). Approximately 1.5 μg of proteins were loaded on a capillary-based Western blot system (ProteinSimple, San Jose, CA, USA) and automatically measured each protein level. Finally, each Western band images were acquired and intensities of the target protein with specific molecular size were quantified by Compass for Simple Western v. 6.1.0. (ProteinSimple). Monoclonal antibodies against B-cell lymphoma 2 (Bcl-2; clone 100; BioLegend), phosphorylated protein kinase B (p-Akt; clone D9E; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:100), Akt (clone 11E7; Cell Signaling Technology, Inc.; 1:100), Sex determining region Y-box 2 (Sox-2; clone D6D9; Cell Signaling Technology, Inc.; 1:100), nuclear factor erythroid-2 related factor 2 (Nrf2; clone D1Z9C; Cell Signaling Technology, Inc.; 1:100), Kelch-like ECH-associated protein 1 (Keap1; clone D6B12; Cell Signaling Technology, Inc.; 1:100), and polyclonal antibodies against β-actin (Cell Signaling Technology, Inc.; 1:200) were used for the analysis.
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!