Detection of phosphorylated Akt and MAPK in cell culture assays.

MethodsX (2016-06-09)
Simon Molgaard, Maj Ulrichsen, Ditte Olsen, Simon Glerup
ABSTRACT

This article describes an immunocytochemistry (ICC) method for staining against phosphorylated forms of the kinases Akt (pAkt) and MAPK (pMAPK). Phosphorylation is induced upon their activation by a number stimuli including insulin and brain-derived neurotrophic factor (BDNF), and is prerequisite for a number of cellular processes including cell proliferation and survival [1], [2], [3], [4], [5], [6]. ICC using antibodies raised against specific phosphorylation sites allows cell-type specific and subcellular monitoring of kinase activation. Here, we test how four different antibodies against pAkt and pMAPK, respectively perform in different cell types following insulin or BDNF stimulation using different protocol conditions. We find that phospho-specific-antibodies generally perform better when using Triton X-100 as a permeabilization agent compared to Saponin. In addition, two antibodies against pAkt and two against pMAPK gave a clear increase in signal in cells stimulated with insulin or BDNF compared to the signal obtained in unstimulated cells. These antibodies also performed well when tested with western blotting. Our results illustrate that both the choice of antibody as well as protocol details are critical parameters for successful detection of phosphorylated forms of kinases by ICC. This article includes: •A protocol for subcellular detection of phosphorylated Akt and MAPK.•Validation of 8 antibodies by immunocytochemistry.•Confirmation by western blotting.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bicinchoninic Acid Kit for Protein Determination, for 200-1000 μg/ml protein
Sigma-Aldrich
TruPAGE Tris-MOPS SDS Express Running Buffer, 20 ×
Sigma-Aldrich
TruPAGE Precast Gels, 4-20%, 10 x 10cm, 12-well

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