Xenopus and Drosophila Cell Extraction

Xenopus CSF and interphase extracts were prepared as described (Castilho et al., 2009 (link)). HeLa cells were extracted in ice-cold phosphatase buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.25% NP-40) according to Singh et al. (2010) (link), or M-PER mammalian cell lysis buffer (Thermo Scientific, Rockford, IL) with the same results. A 1:100 dilution of EDTA-free Proteoblock protease inhibitor (Thermo Scientific) was added to both extraction buffers. Drosophila whole larvae or larval brains were homogenized with a pestle in phosphatase buffer as above, to which was added a 1:100 dilution of phenylmethanesulfonyl fluoride (PMSF; Thermo Scientific), a 1:50 dilution of Halt protease inhibitor cocktail (Thermo Scientific), and a 1:100 dilution of a 100 mg/ml stock solution of soybean trypsin inhibitor (AMRESCO, Solon, OH; catalog number M191).

Free full text: Click here

Williams B.C., Filter J.J., Blake-Hodek K.A., Wadzinski B.E., Fuda N.J., Shalloway D, & Goldberg M.L. (2014). Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers. eLife, 3, e01695.

Publication 2014

phenylmethanesulfonyl fluoride (PMSF; Halt protease inhibitor cocktail soybean trypsin inhibitor

Brains Buffer Cell Cold Dilution Drosophila Edta Egta Hela cells Interphase Larval Mammalian Nacl Np 40 Phenylmethanesulfonyl fluoride Phosphatase Protease inhibitor Solon Soybean Tris Trypsin inhibitor Xenopus

Corresponding Organization :

Other organizations : Cornell University, Vanderbilt University Medical Center

Top 5 similar protocols

Variable analysis

independent variables

Extraction method (phosphatase buffer or M-PER mammalian cell lysis buffer) for HeLa cells

dependent variables

Protein extract from Xenopus CSF and interphase extracts
Protein extract from HeLa cells
Protein extract from Drosophila whole larvae or larval brains

control variables

Phosphatase buffer composition (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.25% NP-40)
EDTA-free Proteoblock protease inhibitor (1:100 dilution) added to both extraction buffers
Phenylmethanesulfonyl fluoride (PMSF; 1:100 dilution), Halt protease inhibitor cocktail (1:50 dilution), and soybean trypsin inhibitor (1:100 dilution of a 100 mg/ml stock solution) added to Drosophila sample preparation

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!