Nature Methods Application Notes e-UPDATE: 12 October 2011

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12 OCTOBER 2011  Application Notes bring you the latest information about innovative tools and technologies and their applications in the lab. We hope that you will find this service useful and informative and encourage you to sign up for future Updates to ensure that you never miss one!

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FEATURED APPLICATION NOTE Identification and Characterization of Key Chemical Constituents for the Authentication of Hoodia Gordonii www.waters.com > Rapidly characterize key chemical constituents from Hoodia Gordonii using UPLC®/oaTof MSE coupled with Multi Variate Statistical Analysis. This method provides a practical workflow to authenticate dietary supplements that claim to contain H.Gordonii. The result is specific data that contains identified key markers for H.Gordonii so that authentication of commercial products, or their herbal extracts, is easily accomplished with confidence. PDF

		EndoLISA®: a novel and reliable method for endotoxin detection www.hyglos.de > A new test for the sensitive detection of endotoxin has been developed, based on a lipopolysaccharide-selective, precoated microplate and a factor C–based detection reagent and presented in a complete kit format. The selective capture of lipopolysaccharide (LPS) is achieved using a phage-derived receptor protein exhibiting high affinity and high specificity for the conserved core region of LPS. After binding of sample-LPS to the microplate as the first stage of the assay, the original sample matrix is washed off, thereby eliminating potentially interfering components. In the second stage of the assay, LPS is detected by factor C in a process whereby the principal receptor of the Limulus amoebocyte coagulation cascade reacts with a fluorescence substrate. The new endotoxin test EndoLISA has a detection range from 0.05 EU/ml up to 500 EU/ml. PDF

		Stellaris™ fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection www.biosearchtech.com > The Stellaris™ FISH technology is a new mRNA detection method that enables simultaneous detection, localization and quantification of individual RNA molecules at the cellular level. This technique is ideal for applications that analyze transcription site activity, protein-RNA interactions and mRNA translocation events, and holds promise for future diagnostic applications. PDF

		Total Internal Reflection Fluorescence (TIRF) Microscopy (TIRFM): A Basic Overview with an Emphasis on the Optics Required www.chroma.com > Total Internal Reflection Fluorescence (Microscopy) is a technique that was developed to restrict the background fluorescence and increase the signal-to-noise ratio (s/n) in the resultant images. This is accomplished in TIRF by using the ability of light to create an evanescent wave (or field) at a very limited range within the sample beyond an interface of two substrates differing in refractive index. In practice, this involves imaging a specimen that is in direct contact with a glass slide or tissue chamber. If the angle of the light is greater than the critical angle, this refractive index mismatch will create a field/wave with properties that are identical in frequency to the light. In other words, a fluorescent molecule that would normally absorb light at 488nm can be 'excited' by the electromagnetic field created by a 488nm laser (or other monochromatic source) that is reflected off of the lower refractive index material. Since this field will decay in intensity exponentially with distance, the resultant fluorescence signal will occur in less than 100 nanometers of the surface. This effectively restricts the excitation to the sample and therefore reduces the z-axis signal which significantly increases the s/n ratio of the sample. Of course, this does mean that the molecules of interest must be within that limited range of the evanescent field. Full Text

		ReliaPrep™ Large Volume HT gDNA Isolation System: gDNA isolation from blood samples www.promega.com > The ReliaPrep™ Large Volume HT gDNA Isolation System enables automated purification of genomic DNA (gDNA) from up to 96 samples of whole blood at a time. The system dramatically increases laboratory throughput while conserving reagents by scaling the purification chemistry automatically to each sample's input volume, providing DNA that is ready for storage or for use in downstream applications without a need to resuspend DNA pellets. PDF

		SUMOpro Gene Fusion Technology II www.lifesensors.com > Expressing and purifying large quantities of functional properly folded protein is a bottleneck in structural and functional genomic studies. Expression difficulties typically include poor yield and formation of insoluble aggregates. These issues compel scientists to use gene fusion technologies to increase protein expression. Gene fusion systems such as glutathione-S-transferase (GST), thioredoxin (Trx), and maltose binding protein (MBP), are hampered by the time-consuming and inefficient process to remove the fusion tag. The proteases used to remove these tags (including thrombin, EK, and TEV protease) recognize short primary sequences, and unfortunately often cleave within the recombinant protein. These protein fusion systems are also hampered by poor protein folding capacity, resulting in formation of improperly folded, inactive proteins that aggregate as insoluble inclusion bodies. PDF

		Tools for Enhancing Sequence Diversity and Reducing Bias in DNA-seq Library Preparation www.biooscientific.com > The generation of high quality next generation sequencing data begins with libraries that have the desired insert size and proper adapter ligation. Artifacts during library preparation can result in PCR duplicates, uneven read coverage and poor adapter ligation efficiencies. We examined this by generating and analyzing DNA sequences from Escherichia coli ge-nomes. Our improved protocol significantly reduces bias, increases ligation efficiency, im-proves indexing flexibility and increases high throughput functionality in DNA libraries pre-pared for sequencing. PDF

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