Cell Fractionation and Western Blotting

Cell fractionation was performed as previously described (Méndez and Stillman, 2000 (link)). Western blots were done on whole-cell lysates as previously described (Bredemeyer et al., 2008 (link)). Anti–NF-κB2 (recognizes p100 and p52; polyclonal), anti-RELB (clone C1E4), anti-BLNK (clone D8R3G), anti-SYK (clone D1I5Q), anti-BTK (clone D3H5), and anti-phospho(S15)-p53 (polyclonal) antibodies were purchased from Cell Signaling Technology. Anti-TRAF2 (polyclonal), anti-TRAF3 (polyclonal), anti-IRF4 (clone E-7), anti-PU.1 (polyclonal), and anti-LaminB (clone G-1) were obtained from Santa Cruz Biotechnology. Anti-phosphorylated BLNK (clone J117-1278) was purchased from BD. Anti-GAPDH (clone GAPDH-71.1) and anti-FLAG (clone M2) were from Sigma-Aldrich. Secondary reagents were horseradish peroxidase (HRP)–conjugated anti–mouse IgG (Cell Signaling Technology) or anti–rabbit IgG (Cell Signaling Technology). Western blots were developed with ECL (Thermo Fisher Scientific) and ECL Prime (GE Healthcare).

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.

Bednarski J.J., Pandey R., Schulte E., White L.S., Chen B.R., Sandoval G.J., Kohyama M., Haldar M., Nickless A., Trott A., Cheng G., Murphy K.M., Bassing C.H., Payton J.E, & Sleckman B.P. (2016). RAG-mediated DNA double-strand breaks activate a cell type–specific checkpoint to inhibit pre–B cell receptor signals. The Journal of Experimental Medicine, 213(2), 209-223.

Publication 2016

anti-phospho(S15)-p53 Anti-GAPDH (clone GAPDH-71.1) anti-FLAG (clone M2) HRP)–conjugated anti–mouse IgG ECL ECL Prime

Anti igg Antibodies Blnk Cell Cell fractionation Clone Gapdh Horseradish peroxidase Irf4 Mouse P100 Rabbit Traf2 Traf3 Western blots

Corresponding Organization : Washington University in St. Louis

Other organizations : University of California, Los Angeles, Children's Hospital of Philadelphia

Top 5 similar protocols

Protocol cited in 2 other protocols

Variable analysis

independent variables

Cell fractionation method (as previously described in Méndez and Stillman, 2000)
Western blot analysis method (as previously described in Bredemeyer et al., 2008)

dependent variables

Levels of NF-κB2 (p100 and p52), RELB, BLNK, SYK, BTK, phospho(S15)-p53, TRAF2, TRAF3, IRF4, PU.1, and GAPDH proteins

control variables

Lamin B (used as a loading control for Western blots)

controls

Positive control: None specified
Negative control: None specified

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.

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?

Revolutionizing how scientists
search and build protocols!