In-Cell Western Protocol for Semi-High-Throughput Screening of Single Clones

Arpita S Pal; Alejandra M Agredo; Andrea L Kasinski

doi:10.21769/BioProtoc.4489

In-Cell Western Protocol for Semi-High-Throughput Screening of Single Clones

Bio Protoc. 2022 Aug 20;12(16):e4489. doi: 10.21769/BioProtoc.4489.

Authors

Arpita S Pal^{1

2}, Alejandra M Agredo^{1

2}, Andrea L Kasinski¹

Affiliations

¹ Department of Biological Sciences, Purdue University, West Lafayette, USA.
² Purdue life Sciences Interdisciplinary Program (PULSe), Purdue University, USA.

Abstract

The in-cell western (ICW) is an immunocytochemical technique that has been used to screen for effects of siRNAs, drugs, and small molecule inhibitors. The reduced time and number of cells required to follow this protocol illustrates its semi-high-throughput nature. Performing a successful ICW protocol requires fixing and permeabilizing adherent cells directly in the plate that specifically exposes the epitope of interest. After blocking of non-specific proteins, the cells are incubated overnight with a primary antibody of interest, which is detected via a host-specific near-infrared fluorescently labeled LI-COR secondary antibody. In the final step, the plate is scanned using an Odyssey LI-COR Imaging System or similar, and each of the wells is quantified. For the first time, this technique has been demonstrated to be reproducibly utilized for semi-high-throughput selection of knockout or overexpression clones. Graphical abstract.

Keywords: CRISPR-Cas9; Doxycycline; High-throughput; In-cell western; Knockout; LI-COR; Overexpression; Screen validation; Single clone selection.