Bench Marks

April’s issue of Cold Spring Harbor Protocols includes instructions for Rapid Coomassie Blue Staining of Protein Gels. This method is an adaptation of the conventional Coomassie staining protocol described in Staining Proteins in Gels with Coomassie Blue. Coomassie Brilliant Blue R250 (CBR-250) is the most commonly used dye for visualizing proteins because of its relatively high sensitivity. The modified method speeds up the destaining process for faster results with increased sensitivity and is compatible with mass-spectrometry-based methods for identifying proteins. Other methods for staining proteins can also be found in Cold Spring Harbor Protocols, including the Zinc/Imidazole Procedure for Visualization of Proteins in Gels by Negative Staining, and Staining Proteins in Gels with Silver Nitrate. Silver Nitrate’s sensitivity is in the low-nanogram range, which is 50-100 times more sensitive than classical Coomassie Blue staining, ~10 times better than colloidal Coomassie Blue staining, and at least twice as sensitive as the zinc/imidazole negative staining method.

The other blog where I write, The Scholarly Kitchen, has been nominated for a Webby, a fairly prestigious award in the online world. Since the winner is determined by the voting public, and since we’re up against some seriously stiff competition (including the NY Times and Wall Street Journal), your help would be greatly appreciated. You can vote for us here. Voting does require you to complete a short registration process. You can only vote once per email address. Voting ends April 29th, and results will be announced May 4th (a visual tutorial on how to vote can be found here).
From the Society for Scholarly Publishing:

On behalf of the SSP leadership, we are very proud that our community has generated a vehicle as widely recognized and effective as the Scholarly Kitchen. Now, we have an opportunity to increase the awareness of the SSP, the Scholarly Kitchen, and scholarly publishing in general.

Thanks in advance for your help!

While 454-based pyrosequencing has led to great advances, an intrinsic artifact of the process leads to artificial over-representation of more than 10% of the original DNA sequencing templates. This is particularly problematic in metagenomic studies, where the abundance of any sequence in a dataset is often used for comparative community analysis. It’s important to remove these artificial replicates before analysis. This phenomenon can skew data interpretation when making comparisons between datasets. As metagenome datasets become more plentiful, the ability to apply more robust statistical tests becomes increasingly important, and the validity of the input datasets becomes more crucial. Tools such as MG-RAST (covered in the January issue of Cold Spring Harbor Protocols in Using the Metagenomics RAST Server (MG-RAST) for Analyzing Shotgun Metagenomes) have the capability to remove exact duplicates, but this captures only a subset of the artificial replicates. In the April issue of Cold Spring Harbor Protocols, Tracy Teal and Thomas Schmidt from Michigan State University present an instruction set for Identifying and Removing Artificial Replicates from 454 Pyrosequencing Data. Their 454 Replicate Filter is a web-based tool that incorporates the algorithm cd-hit. This protocol provides details on how to use the replicate filter and obtain a file of unique sequences for use in metagenomic or transcriptomic analyses. This allows users to obtain a more accurate quantitative representation of the sequence diversity in a dataset.

Neurons are organized into anatomical and functional groups called “circuits”. The activity of these circuits is traditionally monitored using conventional electrophysiological techniques. But some cells, such as the submandibular ganglia, are difficult to impale for intracellular recordings. Instead, viral vectors can be used to deliver fluorescent calcium sensors for detecting activity in a living animal. Calcium Imaging of Neuronal Circuits In Vivo Using a Circuit-Tracing Pseudorabies Virus, from Lynn Enquist and colleagues at Princeton University, provides detailed instructions for the use of the pseudorabies virus (PRV) as a vector for imaging connectivity and activity of neuronal circuits. PRV has a broad host range but does not infect higher-order primates, and it travels along chains of synaptically connected neurons. The PRV strain used in this procedure encodes G-CaMP2, a sensitive fluorescent calcium sensor protein. Available in the April issue of Cold Spring Harbor Protocols, the method allows for reliable detection of endogenous circuit activity at single-cell resolution. As one of April’s featured articles, it is freely available to subscribers and nonsubscribers alike.

The goal of tissue engineering is to recapitulate healthy human organs and tissue structures in culture, and then transplant them into patients, where they are fully integrated. This is a complicated process, and the use of high-throughput imaging systems that allow researchers to directly monitor transplanted tissues in live animals over time is important for improving the culturing and implantation techniques, as well as the design of artificial tissue scaffolds. By using transgenic animals with cell-specific fluorescent reporters, parameters such as tissue perfusion, donor cell survival, and donor-host cell interaction/integration can be observed. In the April issue of Cold Spring Harbor Protocols, Mary Dickinson and colleagues from the Baylor College of Medicine present a protocol for the use of The Mouse Cornea as a Transplantation Site for Live Imaging of Engineered Tissue Constructs. This is a modified version of the classical corneal micropocket angiogenesis assay, which employs it as a live imaging “window” to monitor angiogenic hydrogel tissue constructs. As one of April’s featured articles, it is freely available to subscribers and nonsubscribers alike.