Protocol detail

Protein-Protein Interaction Assay

Find Similar Protocols

Cells were solubilized with lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 50 mM NaF, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF] and centrifuged at 12,000 × g for 20 min. For co-immunoprecipitation assay, cells were sonicated briefly in lysis buffer before centrifugation. The lysates were subjected to immunoprecipitation, SDS-PAGE, and Western blot analyses as described^{2 (link)}.

Free full text: Click here

Nishita M., Park S.Y., Nishio T., Kamizaki K., Wang Z., Tamada K., Takumi T., Hashimoto R., Otani H., Pazour G.J., Hsu V.W, & Minami Y. (2017). Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness. Scientific Reports, 7, 1.

Publication 2017

Aprotinin Assay Buffer Cells Centrifugation Co immunoprecipitation Edta Immunoprecipitation Leupeptin Nacl Np 40 Sds page Tris Western blot analyses

Top 5 similar protocols

Protocol cited in 566 other protocols

Variable analysis

independent variables

Lysis buffer composition (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 50 mM NaF, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.25 mM pAPMSF)
Cell sonication before centrifugation

dependent variables

Co-immunoprecipitation
SDS-PAGE
Western blot analyses

control variables

Centrifugation at 12,000 × g for 20 min

controls

Positive controls not explicitly mentioned
Negative controls not explicitly mentioned

Annotations

Based on most similar protocols

Sonicate samples briefly in lysis buffer before centrifugation to improve cell lysis and protein extraction (Input protocol).

Use ice-cold lysis buffer to maintain protein stability and prevent proteolysis (Protocol 1, Protocol 2, Protocol 5).

Include protease inhibitors such as leupeptin and aprotinin in the lysis buffer to prevent protein degradation (Input protocol, Protocol 1, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

Include phosphatase inhibitors such as sodium orthovanadate and sodium fluoride in the lysis buffer to preserve protein phosphorylation states (Input protocol, Protocol 1, Protocol 2, Protocol 5).

Centrifuge the cell lysates at 12,000 × g for 10-20 minutes to remove cellular debris and insoluble components (Input protocol, Protocol 2, Protocol 5).

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!