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Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography

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Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis

Authors: Koshi Imami and Tomoharu Yasuda, date: 04/20/2019, view: 2694, Q&A: 0

[Abstract] Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling ...

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Authors: Ran Wang and Sumaira Z. Hasnain, date: 07/20/2017, view: 11967, Q&A: 0

[Abstract] Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 10⁶ Da. These large proteins are densely substituted with short glycan chains, which have many important ...

In vitro Assessment of RNA Polymerase I Activity

Author: Marzia Govoni, date: 02/05/2017, view: 5136, Q&A: 0

[Abstract] In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the ...

Acid Extraction of Total Histone from Colon Cancer HCT116 Cells

Authors: Lin-Lin Cao and Wei-Guo Zhu, date: 11/20/2016, view: 6624, Q&A: 0

[Abstract] Histone acid extraction assay is a popular method to determine histone modification levels in mammalian cells. It includes three steps: first, histones are released from chromatin by sulfuric acid; trichloroacetate (TCA) is then added to precipitate histones; and finally, histones are dissolved in double-distilled H₂O (ddH₂ ...

Cell Surface Protein Detection to Assess Receptor Internalization

Authors: Magdalena Czarnecka and Joanna Kitlinska, date: 10/20/2016, view: 6478, Q&A: 0

[Abstract] The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our ...

Preparation of Recombinant Galectin-3 for Cancer Studies

Authors: Kari Tyler, Sok-Hyong Lee and Erwin G. Van Meir, date: 01/05/2016, view: 6373, Q&A: 0

[Abstract] Galectin-3 is a member of a class of proteins termed Galectins, characterized by their ability to bind glycans containing β-galactose (Cummings and Liu, 2009). Galectin-3 binds preferentially to proteoglycans terminating with N-acetyllactosamine (LacNAc) chains (i.e., tandem repeats of galactose) (Newlaczyl and Yu, 2011). Galectin-3 is ...

Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer Cells

Authors: Mike Z Lin, Janet L Martin and Robert C Baxter, date: 05/20/2015, view: 23333, Q&A: 0

[Abstract] Protein-protein interaction networks provide a global picture of cellular function and biological processes, and the dysfunction of some interactions causes many diseases, including cancer. The in situ proximity ligation assay (PLA) is a powerful technology capable of detecting the interactions among proteins in fixed tissue and cell ...

Detection of Phospho-KRAS by Electrophoretic Mobility Change in Human Cell Lines and in Tumor Samples from Nude Mice Grafts

Authors: Carles Barceló, Noelia Paco, Debora Cabot, Eduard Garrido, Neus Agell and Montserrat Jaumot, date: 03/20/2015, view: 7042, Q&A: 0

[Abstract] KRAS is the oncogene most frequently mutated in human solid tumors especially in pancreas, colon, small intestine, biliary tract and lung. We have recently demonstrated that oncogenic KRAS needs S181 phosphorylation to fully display its oncogenic features suggesting its inhibition as a therapeutic treatment against KRAS-driven tumors. Due to the ...

Analysis of Protein Stability by the Cycloheximide Chase Assay

Authors: Shih-Han Kao, Wen-Lung Wang, Chi-Yuan Chen, Yih-Leong Chang, Yi-Ying Wu, Yi-Ting Wang, Shu-Ping Wang, Alexey I Nesvizhskii, Yu-Ju Chen, Tse-Ming Hong and Pan-Chyr Yang, date: 01/05/2015, view: 50485, Q&A: 1

[Abstract] Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a ...

Hypoxia Studies with Pimonidazole in vivo

Authors: Kristina Y. Aguilera and Rolf A. Brekken, date: 10/05/2014, view: 18965, Q&A: 0

[Abstract] Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including ...

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