Western Blot Analysis of T Cell Proteins

T cells (0.6×10⁶) were lysed in (35 μl) Carin lysis buffer (20 mM Tris-HCl [pH 8.0], 138 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% Nonidet P-40, 10% glycerol, 2 mM Na vanadate) supplemented with complete protease inhibitors (Roche). Proteins were separated on 10% SDS-polyacrylamide gels and blotted onto PVDF membranes according to standard protocols. The following primary antibodies were used: anti-actin (AC-74; Sigma-Aldrich, St. Louis, MO), mouse anti-Runx3 (2B3; Abcam) and rabbit anti-Th-POK ^{22 (link)}. Secondary antibodies were a peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) and a HRP-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch Laboratories). Signals were detected using ECL (SuperSignal West Dura Extended Duration Substrate from ThermoScientific).

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.

Boucheron N., Tschismarov R., Goeschl L., Moser M.A., Lagger S., Sakaguchi S., Winter M., Lenz F., Vitko D., Breitwieser F.P., Müller L., Hassan H., Bennett K.L., Colinge J., Schreiner W., Egawa T., Taniuchi I., Matthias P., Seiser C, & Ellmeier W. (2014). CD4+ T cell lineage integrity is controlled by the histone deacetylases HDAC1 and HDAC2. Nature immunology, 15(5), 439-448.

Publication 2014

anti-actin (AC-74; mouse anti-Runx3 (2B3; AffiniPure goat anti-rabbit IgG (H+L) SuperSignal West Dura Extended Duration Substrate

Actin Anti igg Antibodies Buffer Dura Edta Glycerol Goat Membranes Mouse Nacl Nonidet p 40 Peroxidase Polyacrylamide gels Protease inhibitors Proteins Pvdf Rabbit T cells Tris Vanadate

Corresponding Organization :

Other organizations : Medical University of Vienna, Wellcome Centre for Cell Biology, University of Edinburgh, Max Perutz Labs, Vienna Biocenter, CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences, Washington University in St. Louis, RIKEN Center for Integrative Medical Sciences, Novartis (Switzerland), University of Basel, Friedrich Miescher Institute

Top 5 similar protocols

Variable analysis

independent variables

Lysis buffer composition (20 mM Tris-HCl [pH 8.0], 138 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% Nonidet P-40, 10% glycerol, 2 mM Na vanadate)

dependent variables

Protein expression levels of Actin, Runx3, and Th-POK

control variables

T cell count (0.6×10^6)
Lysis buffer volume (35 μl)
Protease inhibitors (Roche complete protease inhibitors)
SDS-PAGE and western blotting protocols

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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!