Authors
Takahiro Mimae, Akihiko Ito, Man Hagiyama, Jun Nakanishi, Yoichiroh Hosokawa, Morihito Okada, Yoshinori Murakami & Tadashi Kondo
Summary
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.Further details
The protocol was published on Protocol Exchange on 4 March 2014. To see the entire protocol, click on the source link.Advertisement
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