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Gallagher S.R.,UVP LLC
Current Protocols in Essential Laboratory Techniques | Year: 2014

This unit is an overviewof the subsequent units on protein and nucleic acid electrophoresis separation techniques. Curr. Protoc. Essential Lab. Tech. 8:7.1.1-7.1.7. © 2014 by John Wiley & Sons, Inc. Source


Gallagher S.R.,UVP LLC
Current Protocols in Essential Laboratory Techniques | Year: 2014

ImageJ is a freely available, cross-platform (e.g., Windows, Mac, Linux) image processing and analysis program developed by the NIH. In addition to being readily available for no cost, ImageJ is supported by a wide range of constantly evolving user-created functionalities to address a remarkable range of applications, complementing commercial software that typically comes with imaging instruments such as digital gel-imaging systems or microscopy workstations. New processing/analysis macros and plug-ins are routinely added to the support site, and are frequently validated via refereed publications. With the continued improvements and growth of fluorescence-based applications, ImageJ continues to be a mainstay in the laboratory. ImageJ has extensive support materials available online, its base code is regularly updated, and a survey of Medline references indicates that it is one of the most widely used image-analysis packages available today. © 2014 by John Wiley & Sons, Inc. Source


Gallagher S.R.,UVP LLC
Current Protocols in Essential Laboratory Techniques | Year: 2012

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), investigate subunit compositions, verify homogeneity of protein samples, and purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Support protocols cover the casting of gels, calculation of molecular mass using the electrophoretic mobility of a protein, and purification of SDS by recrystallization. © 2012 by John Wiley & Sons, Inc. Source


Gallagher S.R.,UVP LLC
Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.] | Year: 2011

Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 μg/ml. Absorbance at 260 nm has an effective range from 1 to 50 μg/ml; Hoechst 33258 from 0.01 to 15 μg/ml; ethidium bromide from 0.1 to 10 μg/ml; and PicoGreen from 25 to 1000 pg/ml. Source


Gallagher S.R.,UVP LLC
Current Protocols in Essential Laboratory Techniques | Year: 2010

Immunoblotting (also referred to as western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by the polyacrylamide gel and, to make the separated proteins accessible, the proteins need to be moved out of the gel and bound to a rectangular sheet of PVDF or nitrocellulose membrane. Specialized blotting equipment electrophoretically transfers the negatively charged proteins from the gel onto the membrane. The nitrocellulose or PVDF membrane binds the proteins as they move out of the gel, producing an exact replica, on the membrane surface, of the original protein gel separation. The membrane is then blocked to prevent any nonspecific protein binding and visualized by specific antibodies to detect the presence or absence of a particular protein. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression, and post-translational modification analysis. © 2010 by John Wiley & Sons, Inc. Source

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