Mar 25, 2025
Variant painting via immunostaining
- 1University of Toronto
Protocol Citation: Chloe Reno 2025. Variant painting via immunostaining. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3qmepv25/v1
Manuscript citation:
Jessica Lacoste, Marzieh Haghighi, Shahan Haider, Chloe Reno, Zhen-Yuan Lin, Dmitri Segal, Wesley Wei Qian, Xueting Xiong, Tanisha Teelucksingh, Esteban Miglietta, Hamdah Shafqat-Abbasi, Pearl V. Ryder, Rebecca Senft, Beth A. Cimini, Ryan R. Murray, Chantal Nyirakanani, Tong Hao, Gregory G. McClain, Frederick P. Roth, Michael A. Calderwood, David E. Hill, Marc Vidal, S. Stephen Yi, Nidhi Sahni, Jian Peng, Anne-Claude Gingras, Shantanu Singh, Anne E. Carpenter, Mikko Taipale,
Pervasive mislocalization of pathogenic coding variants underlying human disorders,
Cell,
Volume 187, Issue 23,
2024,
Pages 6725-6741.e13,
ISSN 0092-8674,
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 24, 2025
Last Modified: March 25, 2025
Protocol Integer ID: 123268
Funders Acknowledgements:
NIH IGVF
Grant ID: HG011989
Abstract
High-throughput imaging platform to assay the impact of coding variation on protein localization. Proteins are flag-tagged and immunostained for visualization, alongside additional cellular compartments including the nucleus, mitochondria, and ER.
Day 1: Cell Seeding
Day 1: Cell Seeding
Seed 4,000-5,000 HeLa Kyoto Cells - 96-well format, 100 µL per well. Media is high glucose DMEM supplemented with 10% FBS, 1% penicillin-streptomycin. Incubate Overnight at 37 °C , 5% CO2
Day 2: Transfection
Day 2: Transfection
2d 0h 15m
2d 0h 15m
Prepare Transfection Complex Master Mix in 96-well v-bottom plates
Note
First check cells to make sure they are at optimal (50-60%) confluency for transfection.
Per well, mix:
1. 10 uL of diluted DNA (200 ng total)
Note
Add DNA to wells first. Prepare a master mix of OptiMEM + X-tremeGENE 9. Vortex to mix. Make extra master mix to account for error.
2. 0.3 uL X-tremeGENE 9
3. 30 uL OptiMEM
Let stand at room temperature for 00:15:00
15m
Mix by pipetting up and down. Transfer transfection complex to seeded HeLa cells dropwise.
Incubate for 48:00:00 at 37 °C , 5% CO2
2d
Day 4: Fixation & Staining
Day 4: Fixation & Staining
3h 55m
3h 55m
Stain live cells with Mitotracker:
Make 1mM stock of tracker dye fresh. Before opening, allow the vial to warm to room temperature and then briefly centrifuge the vial.
Note
For Mitotracker Red – add 94.1 µL DMSO to vial.
Dilute the 1 mM stock solution to the final working concentration (100 nM for Mito, 1 µM for Lysotracker) in growth medium (1xDMEM complete).
Add to cells and incubate for 01:00:00 in the tissue culture incubator.
1h
Wash cells 3 times with 1xPBS.
Fix cells
Add 30 uL/well of 4% paraformaldehyde diluted in complete DMEM
Wrap the plate in foil. Incubate for 00:15:00 at room temp
15m
Wash cells 3 times with 1xPBS.
Complete intracellular staining:
Incubate with 50µL/well of 1xPBS/0.1% Triton X-100 for 00:10:00 , room temperature
shaking gently for permeabilization.
10m
Incubate with 50µL/well of 1xPBS/0.1% Triton X-100/1% BSA for 00:30:00 , room temperature shaking gently for blocking.
30m
Incubate with primary antibody of choice diluted in 30µL/well blocking buffer for 01:00:00 , room temperature shaking gently (For anti-FLAG, 1:500).
1h
Wash three times with 1xPBS.
Dilute the secondary antibodies (anti-mouse 488, 1:500; Hoechst, 1:5000; Concanavalin
A, 1:250) in 30 µL/well blocking buffer. Incubate for 01:00:00 at room temperature
shaking gently.
1h
Wash three times with 1xPBS.
Leave in 50µL 1xPBS for imaging.