Protocols in Current Issue
    Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
    Authors:  Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 1205, Q&A: 0
    [Abstract] Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have ...
    Virulence Studies of Clostridium difficile
    Authors:  Michael B. Francis and Joseph A. Sorg, date: 12/20/2013, view: 3506, Q&A: 0
    [Abstract] Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) can colonize antibiotic-treated hosts (McFarland, 2008). Antibiotics alter the composition of the normal, benign microbial flora which leads to loss of colonization resistance (Wilson and Perini, 1988; Antonopoulos et al., 2009). C. difficile spores ...
    pERK Detection Assays Using the Surefire AlphaScreen® Kit (TGR Biosciences and PerkinElmer)
    Authors:  Cassandra Koole, Denise Wootten and Patrick Sexton, date: 07/05/2013, view: 4379, Q&A: 0
    [Abstract] Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are serine/threonine protein kinases that are phosphorylated on Thr202/Tyr204 (ERK1) and Thr185/Tyr187 (ERK2). Phosphorylation of ERK1/2 (pERK1/2) arises from multiple stimuli, resulting in physiological responses that include cell growth, proliferation and differentiation. This protocol has ...
    cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer)
    Authors:  Cassandra Koole, Denise Wootten and Patrick Sexton, date: 07/05/2013, view: 5769, Q&A: 0
    [Abstract] Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ...
    Measurement of Free Cytosolic Calcium Concentration ([Ca2+]i) in Single CHO-K1 Cells
    Authors:  Manuel D. Gahete, Raúl M. Luque and Justo P. Castaño, date: 11/20/2012, view: 5895, Q&A: 0
    [Abstract] This is a protocol to analyze the functional response of single CHO-K1 cells to a given treatment in terms of changes in free cytosolic calcium concentration ([Ca2+]i). This is possible by using the Ca2+ indicator dye Fura-2 AM, a polyamino carboxylic acid that binds to free intracellular calcium and is excited at 340 nm and 380 nm. The ratio of ...