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

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Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation

Authors: Abhilash Padavannil, Chad A. Brautigam and Yuh Min Chook, date: 05/20/2020, view: 125, Q&A: 0

[Abstract] Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the ...

Fibrin Breakdown Assay

Authors: Jiayue Ling, Connor M. Blair and George S. Baillie, date: 04/20/2020, view: 813, Q&A: 0

[Abstract] Fibrinolysis is an integral part of the matrix remodeling process that contributes to tissue repair. Fibrin clots are broken down during fibrinolysis in a controlled process. Fibrin degradation products (FDPs) have also been shown to have a role in the regulation of cell growth and are implicated in various vascular diseases. This protocol was ...

Quantitative Determination of Ca²⁺-binding to Ca²⁺-sensor Proteins by Isothermal Titration Calorimetry

Authors: Seher Abbas and Karl-Wilhelm Koch, date: 04/05/2020, view: 384, Q&A: 0

[Abstract] Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from ...

Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method

Authors: Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 438, Q&A: 0

[Abstract] This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (E_m = -55 mV) in the SdhA flavoprotein subunit of succinate ...

Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry

Authors: Claudia Cattoglio, Xavier Darzacq, Robert Tjian and Anders Sejr Hansen, date: 02/20/2020, view: 1226, Q&A: 0

[Abstract] Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (e.g., fluorescence ...

Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces

Authors: Roberta M Sabino and Ketul C Popat, date: 02/05/2020, view: 784, Q&A: 0

[Abstract] Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. ...

Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis: An Integrated Protocol

Authors: Alexandra R Crowe and Wei Yue, date: 12/20/2019, view: 1498, Q&A: 0

[Abstract] Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed ...

Separation of Natural Collagen Crosslinks Using Buffer and Ion-pairing Agent Free Solvents on Silica Hydride Column for Mass Spectrometry Detection

Authors: Rafea Naffa and Joseph Pesek, date: 05/05/2019, view: 1403, Q&A: 0

[Abstract] In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation ...

Measurement of Redox States of the β3 Integrin Disulfide Bonds by Mass Spectrometry

Author: Joyce Chiu, date: 02/05/2019, view: 1885, Q&A: 0

[Abstract] Functional disulfide bonds mediate a change in protein function in which they reside when cleaved or formed. To elucidate how a functional disulfide bond controls protein activity, it is critical that the redox state of the bond in the population of protein molecules is known. Measurement of changes in disulfide bond redox state relies on thiol ...

Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages

Authors: Gaurav Kumar, Subhash B. Arya and Amit Tuli, date: 12/20/2018, view: 2459, Q&A: 0

[Abstract] Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs ...

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