Using chemiluminescence imaging of cells (CLIC) for relative protein quantification
Fisher, Jane LU ; Sørensen, Ole E. LU and Abu-Humaidan, Anas H.A. LU
(2020)
In Scientific Reports
10(1).
- Abstract
Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding... (More)
Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells.
(Less)
- author
- Fisher, Jane
LU
; Sørensen, Ole E.
LU
and Abu-Humaidan, Anas H.A.
LU
- organization
- publishing date
- 2020
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Scientific Reports
- volume
- 10
- issue
- 1
- article number
- 18280
- publisher
- Nature Publishing Group
- external identifiers
-
- scopus:85093846359
- pmid:33106566
- ISSN
- 2045-2322
- DOI
- 10.1038/s41598-020-75208-0
- language
- English
- LU publication?
- yes
- id
- acdd8bdb-019f-4c9a-ba48-d40163f5d9ae
- date added to LUP
- 2020-11-04 13:37:10
- date last changed
- 2024-09-19 09:15:23
@article{acdd8bdb-019f-4c9a-ba48-d40163f5d9ae,
abstract = {{<p>Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells.</p>}},
author = {{Fisher, Jane and Sørensen, Ole E. and Abu-Humaidan, Anas H.A.}},
issn = {{2045-2322}},
language = {{eng}},
number = {{1}},
publisher = {{Nature Publishing Group}},
series = {{Scientific Reports}},
title = {{Using chemiluminescence imaging of cells (CLIC) for relative protein quantification}},
url = {{http://dx.doi.org/10.1038/s41598-020-75208-0}},
doi = {{10.1038/s41598-020-75208-0}},
volume = {{10}},
year = {{2020}},
}