This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
A novel approach to pseudopodia proteomics: excimer laser etching, two-dimensional difference gel electrophoresis, and confocal imaging
Tadashi Kondo
Division of Pharmacoproteomics, National Cancer Center Research Institute
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Pseudopodia are actin-rich ventral cellular protrusions shown to facilitate the migration and metastasis of tumor cells. Here, we present a novel approach to perform pseudopodia proteomics. Tumor cells growing on porous membranes extend pseudopodia into the membrane pores. In our method, cell bodies are removed by horizontal ablation at the basal cell surface with the excimer laser while pseudopodia are left in the membrane pores. For protein expression profiling, whole cell and pseudopodia proteins are extracted with a lysis buffer, labeled with highly sensitive fluorescent dyes, and separated by two-dimensional gel electrophoresis. Proteins with unique expression patterns in pseudopodia are identified by mass spectrometry. The effects of the identified proteins on pseudopodia formation are evaluated by measuring the pseudopodia length in cancer cells with genetically modified expression of target proteins using confocal imaging. This protocol allows global identification of pseudopodia proteins and evaluation of their functional significance in pseudopodia formation within one month.
Keywords
pseudopodia; proteomics; excimer laser ablation; two-dimensional difference gel electrophoresis; confocal imaging
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.mpg
Supplementary Video 1 Image of the excimer laser scanning of the cells cultured on the porous membrane <iframe width="480" height="360" src="//www.youtube.com/embed/FvxJF6fVFgw?rel=0" frameborder="0" allowfullscreen></iframe>
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 04 Mar, 2014
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Associated Publications
Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis
by Akihiko Ito, Takahiro Mimae, Ying-Shan-Zhu Yamamoto, +7
Laboratory Investigation (04 March, 2014)
α-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma
by Masaoki Ito, Man Hagiyama, Takahiro Mimae, +7
Breast Cancer Research And Treatment (04 March, 2014)
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
A novel approach to pseudopodia proteomics: excimer laser etching, two-dimensional difference gel electrophoresis, and confocal imaging
Tadashi Kondo
Division of Pharmacoproteomics, National Cancer Center Research Institute
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 04 Mar, 2014
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Pseudopodia are actin-rich ventral cellular protrusions shown to facilitate the migration and metastasis of tumor cells. Here, we present a novel approach to perform pseudopodia proteomics. Tumor cells growing on porous membranes extend pseudopodia into the membrane pores. In our method, cell bodies are removed by horizontal ablation at the basal cell surface with the excimer laser while pseudopodia are left in the membrane pores. For protein expression profiling, whole cell and pseudopodia proteins are extracted with a lysis buffer, labeled with highly sensitive fluorescent dyes, and separated by two-dimensional gel electrophoresis. Proteins with unique expression patterns in pseudopodia are identified by mass spectrometry. The effects of the identified proteins on pseudopodia formation are evaluated by measuring the pseudopodia length in cancer cells with genetically modified expression of target proteins using confocal imaging. This protocol allows global identification of pseudopodia proteins and evaluation of their functional significance in pseudopodia formation within one month.
Figure 1
Figure 2
Figure 3
Associated Publications
Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis
by Akihiko Ito, Takahiro Mimae, Ying-Shan-Zhu Yamamoto, +7
Laboratory Investigation (04 March, 2014)
α-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma
by Masaoki Ito, Man Hagiyama, Takahiro Mimae, +7
Breast Cancer Research And Treatment (04 March, 2014)