Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Solid-phase binding assay

VK Vardit Kram
TK Tina M. Kilts
NB Nisan Bhattacharyya
LL Li Li
MY Marian F. Young
request Request a Protocol
ask Ask a question
Favorite

Bait proteins were bound to either Pierce® Nickel coated plates (15242; Thermo scientific) or ANTI-FLAG® high sensitivity, M2 coated plates (P2983, Sigma) by 2 h incubation at 37 °C. Unbound protein was removed by repeated washing and non-specific binding sites were blocked with 0.1–1% BSA/TBS. Bait-coated plates were incubated with prey proteins at 4 °C overnight. Binding of the prey protein to the coated plate was detected using primary antibodies raised against the prey proteins, combined with species matched HRP-conjugated secondary antibodies. Plates were read at 450 nm after HRP chromogenic substrate reaction using the TMB-peroxidase substrate system (KPL, Gaithersburg, MD, USA) according to manufacturer’s protocol. List of proteins and primary antibodies used can be found in Tables S2 and S3.

Copyright and License information: The Author(s) ©2017
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A