In Vitro Phosphorylation of LCN2 by PKCδ

In vitro phosphorylation of purified LCN2 by recombinant PKCδ (Invitrogen) was performed as described [22 (link)]. The kinase reaction was initiated at 37°C by adding recombinant LCN2 and [γ-³²P]ATP. At different time points, the reaction mixture was stopped by the addition of SDS sample buffer. Proteins were separated on NuPAGE Bis-Tris gels (Invitrogen) and stained using SimplyBlue (Invitrogen). Phosphorylated LCN2 was detected by phosphorimaging (Typhoon 9410, Amersham Bioscience).

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Weng Y.C., Wang G., Messing R.O, & Chou W.H. (2015). Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils. Journal of Biomedical Science, 22(1), 21.

Publication 2015

NuPAGE Bis-Tris gels SimplyBlue Typhoon 9410

Bis tris Buffer Gels Kinase Phosphorylation Proteins Typhoon

Corresponding Organization :

Other organizations : Kent State University, University of California, San Francisco

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Variable analysis

independent variables

Recombinant PKCδ (Invitrogen)

dependent variables

Phosphorylation of purified LCN2

control variables

Reaction temperature at 37°C
SDS sample buffer used to stop the reaction
NuPAGE Bis-Tris gels (Invitrogen) used for protein separation
SimplyBlue (Invitrogen) used for protein staining
Phosphorylated LCN2 detected by phosphorimaging (Typhoon 9410, Amersham Bioscience)

controls

Positive control: Recombinant LCN2 and [γ-32P]ATP
Negative control: Not explicitly mentioned

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