Cardiomyocyte Protein Fractionation and Analysis

Cardiomyocytes were washed twice with cold 1X PBS and lysed with RIPA buffer (Sigma) containing 1X mammalian protease inhibitor (Sigma, P8340). Proteins in cell homogenates were separated on SDS-PAGE and transferred to nitrocellulose membrane (LI-COR). Soluble and insoluble protein fractions were prepared as reported before^{68 (link)}. Antibodies against Troponin I (Millipore), Nef (NIH), LC3-II (Sigma), Rab7 (Cell Signaling), p62 (Cell signaling), Beclin 1 (Cell Signaling, 3495) Akt (Cell Signaling, 9272) and pAkt (Cell Signaling), GFP (Clontech), Actinin (Sigma) were used for the Western blot analysis.

Free full text: Click here

Gupta M.K., Kaminski R., Mullen B., Gordon J., Burdo T.H., Cheung J.Y., Feldman A.M., Madesh M, & Khalili K. (2017). HIV-1 Nef-induced cardiotoxicity through dysregulation of autophagy. Scientific Reports, 7, 8572.

Publication 2017

RIPA buffer mammalian protease inhibitor nitrocellulose membrane Troponin I LC3-II Beclin 1 pAkt

Actinin Antibodies Beclin 1 Buffer Cardiomyocytes Cell Cold Mammalian Membrane Nitrocellulose Protease inhibitor Protein Ripa Sds page Troponin i Western blot analysis

Corresponding Organization :

Other organizations : Temple University

Top 5 similar protocols

Variable analysis

independent variables

Washing cardiomyocytes twice with cold 1X PBS
Lysing cells with RIPA buffer containing 1X mammalian protease inhibitor

dependent variables

Protein expression levels of Troponin I, Nef, LC3-II, Rab7, p62, Beclin 1, Akt, pAkt, GFP, and Actinin

control variables

Preparation of soluble and insoluble protein fractions as reported before
Use of specific antibodies for Western blot analysis

positive controls

Not explicitly mentioned

negative controls

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!