Tryptic digestion of samples and subsequent analysis by mass spectrometry is a popular technique for the identification of proteins typically those related to interaction partners or biomarkers characterization. This powerful tool can also be used for less traditional experimental designs. Three examples are:
Continue reading “Trypsin: Innovative Applications”Undoubtedly, the polymerase chain reaction (PCR) has revolutionized biological research and has become one of the most common techniques in today’s laboratory. At times, it seems that a new variation of PCR is described in the literature every month. You might think that you are familiar with the dozens of PCR variations, but I am guessing that you haven’t heard of some of these.
Cell-free protein synthesis has emerged as powerful alternative to cell based protein expression for functional and structural proteomics. The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptionaland translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression (1).
Continue reading “Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein”
The use of reporter genes for simple analysis of promoter activity (promoter bashing) is a well known practice. However, there are many other elegant applications of reporter technologies. One such application is illustrated in the paper by Zheng et al., published in the Sept. 2008 issue of Cancer Research. These researchers from the Hormel Institute at the University of Minnesota showed that the cyclin-dependent kinase cdk3 phosphorylates the transcription factor ATF1 and enhances its transcriptional and transactivation activity. The observed cdk/ATF1 signaling was shown to have an important role in cell proliferation and transformation. To do this they used several variations of a reporter-based two-hybrid assay.
Continue reading “Variations on the Two-Hybrid Assay”
Life is complicated. So is death. And when the cells in your multiwell plate die after compound treatment, it’s not enough to know that they died. You need to know how they died: apoptosis or necrosis? Or, have you really just reduced viability, rather than induced death? Is the cytotoxicity you see dose-dependent? If you look earlier during drug treatment of your cells, do you see markers of apoptosis? If you wait longer, do you observe necrosis? If you reduce the dosage of your test compound, is it still cytotoxic? Continue reading “Describing Life and Death in the Cell”
As part of my job I occasionally search the literature for papers citing use of Promega products in new or interesting ways. Any search on dual-luciferase reporters usually generates a lot of returns. A search for dual-luciferase on Highwire press generates over 700 articles from 2009 alone. So why are dual-luciferase reporter assays so widely used? Continue reading “Why Two Reporters are Better than One”
Cell-free protein synthesis (aka: in vitro translation) refers to protein production in vitro using lysates that provide the cellular machinery necessary for synthesis. Ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation/elongation/termination factors, GTP, ATP, Mg2+ and K+ are among the requirements for a translation system. These are provided by lysates, which can be from prokaryotic or eukaryotic sources, depending on your requirements.
Cell-free protein synthesis is most commonly used for generating protein for study of things like:
10. Modify reaction buffer composition to adjust pH and salt concentration.
Continue reading “Top Ten Things You Can Do to Improve Your PCR Results”
