Cell Biology

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    Protocols in Current Issue
    Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
    Authors:  Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 1192, Q&A: 0
    [Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...
    Phospho-protein Analysis in Adherent Cells Using Flow Cytometry
    Authors:  Renu Sharma, Amit Sharma, Atul Kumar and Bithiah Grace Jaganathan, date: 10/20/2019, view: 2093, Q&A: 0
    [Abstract] Protein phosphorylation is one of the most important post-translational modifications, which acts as a reversible on or off switch for the activity of a large number of proteins. Analyzing the phosphorylation status of different proteins can reveal the alterations in the state of the cells in response to cellular damage, cancer and pharmaceutical ...
    Non-radioactive LATS in vitro Kinase Assay
    Authors:  Audrey W. Hong and Kun-Liang Guan, date: 07/20/2017, view: 8203, Q&A: 1
    [Abstract] This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate.
    Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation
    Authors:  Go R. Sato, Tatsuma Ban and Tomohiko Tamura, date: 05/20/2017, view: 9909, Q&A: 0
    [Abstract] While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where ...
    In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
    Authors:  Yuuki Sakai, Shin-ichiro Inoue and Shingo Takagi, date: 11/05/2015, view: 6298, Q&A: 0
    [Abstract] An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the ...
    Substrate Specificity of Recombinant Ser/Thr Protein Kinase
    Authors:  Anna A. Zorina, Galina V. Novikova and Dmitry A. Los, date: 03/20/2015, view: 7444, Q&A: 0
    [Abstract] Protein kinases are enzymes that phosphorylate proteins in a cell. Determination of kinase activity in reactions of phosphorylation is a very convenient way for a biochemical characterization of this group of enzymes. Here we describe a method to determine the activity of a recombinant Ser/Thr protein kinase using as a possible substrate MBP, H1, ...
    TGFβ Stimulation Assay
    Author:  Emanuel Rognoni, date: 12/05/2014, view: 11753, Q&A: 0
    [Abstract] TGFβ is part of a growth factor superfamily which modulates cell growth, differentiation, adhesion, migration, ECM synthesis and apoptosis (Massague, 1998; Siegel and Massague, 2003). Free TGFβ binds to its high affinity TGFβ receptor, a receptor serine/threonine kinase, inducing phosphorylation of Smad2/3 which subsequently forms a complex with ...
    IP-Kinase Assay
    Author:  Pearl A. Campbell, date: 03/05/2014, view: 17535, Q&A: 0
    [Abstract] Immunoprecipitation (IP)- Kinase assays are an invaluable tool to assess the activation status of intracellular signaling cascades within a specific cellular state and also to confirm the enzymatic activity of a specific kinase towards a putative substrate of interest. Intracellular signal transduction cascades play an important role in ...
    Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro
    Authors:  Qing Chen, Alice Boulanger, Deborah M. Hinton and Scott Stibitz, date: 11/20/2013, view: 8920, Q&A: 0
    [Abstract] Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of the Bordetella pertussis response regulator BvgA, which is phosphorylated at an ...
    MAPK Phosphorylation Assay with Leaf Disks of Arabidopsis
    Authors:  Pascale Flury, Dominik Klauser, Thomas Boller and Sebastian Bartels, date: 10/05/2013, view: 9920, Q&A: 0
    [Abstract] Activation of mitogen activated protein kinases (MAPKs) is involved in many abiotic and biotic stress responses including plant defense. MAPK acitvation is based on the dual phosphorylation of threonine (T) and tyrosin (Y) residues (T-x-Y motif) in the activation loop of the MAPK protein. By determination of the phosphorylation status of a ...



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