We are used to seeing multicolored fluorescence images labeling either specific events or structures within cells. When compared to imaging with fluorescent methods, bioluminescence imaging methods provide the advantages of low background and subsequent higher signal to noise ratio—enhancing sensitivity. A key prerequisite for dual-imaging experiments is the ability to distinguish the signal from each event separately and clearly. However, compared to the large number of available fluorescent compounds (many spectrally distinct fluorescent proteins and dyes), there are not many different luciferases to choose from. This lack of variety has limited the capabilities of bioluminescence for imaging multiple molecular events in the same sample. Therefore, there is a need for new luciferases with substrates and emission spectra that are different from the beetle luciferases currently in widespread use.
A paper published in Molecular Imaging in October 2013 describes use of firefly and the new NanoLuc® Luciferase to image cell signaling events in cultured cells and in a mouse model system. The paper, authored by Stacer et al. of the University of Michigan, details a proof-of-concept experiment using firefly and NanoLuc luciferases to image two distinct events in the TGF-beta1 signaling pathway. Continue reading
When I was characterizing proteins in graduate school, my life was filled with constructs, constructs, constructs. I made a variety of subclones to synthesize and isolate parts and pieces of the protein in vitro. I made clones and subclones to generate a panel of antibodies against different parts of the protein. Some of those antibodies ended up working best on Westerns; others performed better in immunocytochemistry experiments. There was no one tool or tag that could be used for every step in the characterization of the protein.
Halotag® fusion tag changes that.
The HaloTag® fusion tag spans both worlds of looking at proteins in isolation for studies of protein interactions and post-translational modifications to studying proteins in cells through real-time imaging or localization studies. Continue reading
Synthesizing proteins in vitro through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function. If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale). There are many systems out there, and knowing which to use can sometimes be difficult. Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA. But when you want to make your own RNA templates to add to lysates, then there are additional concerns.
Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason. Extracting it requires the utmost care in technique and elimination of nucleases. Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease). Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.
The first step for using cell-free expression systems with RNA templates is to make the RNA. Here are some tips that will ensure success. Continue reading
The new NanoLuc® Luciferase is a very small, very bright luciferase, making it ideal when you need a genetic reporter to act near physiological levels inside cells to reveal subtle regulatory events. In the chalk talk below, we illustrate why this is important with a p53/mdm2 example.
When searching for a job it’s important to consider the job duties as well as the company and the company’s culture. Two companies have become famous for their cultures—Google and Zappos. Google is known as a company where you work hard in an amazing environment. Oh, and the food is free! Zappos is known as a place where employees are valued, and customer service is the first priority. Here at Promega, science rules, employee well-being is extremely important, and you can make a big impact regardless of your job title.
If you are able to find a company with an appealing culture and similar values to your own, it is a win-win situation. You will likely be happier in your job and therefore a better performer.
Here are some questions that you can ask to learn about the company culture and figure out if it is a fit with your personality and needs. These questions can be asked in an interview or in an informational conversation with someone in your network before you apply for a job. Keep in mind that there is no right answer to these questions. Some people thrive in government jobs while others have more of an entrepreneurial spirit; you need to figure out what type of culture will work best for you. Continue reading
Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for in-solution or in-gel digestion reactions.
The following references illustrate the use of Asp-N in recent publications:
Protein sequence coverage
- Jakobsson, M et al. (2013) Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
- Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I. J. Biol. Chem. 288, 24799–08.
- Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
- Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot. 12, 932–44.
- Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”. J. Biol. Chem. 288, 30192–09.
- Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot. 12, 3453–64.
This review is a guest blog by Amy Landreman, Product Specialist in Cellular Analysis at Promega Corporation.
Lentiviral vectors (LVV) have become a valuable research tool for delivering genetic content into a wide range of cell types. Commonly derived from the HIV-1 genome, LVV have the advantage of being able to infect both dividing and non-dividing cells. They can be particularly valuable for introducing genetic material into cell lines that are difficult to transfect using other methods and are also being used in gene therapy applications.
Unlike other gene delivery tools, transducing mammalian cells with LVV requires significant upfront effort since the LVV particles carrying the desired genetic content first need to be created. In general this involves co-transfecting a packaging cell line, such as HEK293T, with a set of three to four separate plasmids that encode the protein content required to generate the LVV particles: the transfer plasmid, which contains the transgene of interest, a packaging plasmid, and an envelope plasmid. After co-transfection, the packaging cell line is allowed to incubate for a couple of days during which time the LVV particles are produced and accumulating in the culture supernatant. The supernatant containing the recombinant LVV is then harvested and, following several concentration steps, the LVV particles are ready to be used for introducing the desired genetic content into the mammalian target cells. Continue reading
When my son was about 2 years old, he commented that the jingles “Twinkle twinkle little star” and “alphabet song” had the same musical notation. While I do not think I am tone deaf and I do appreciate music, I had not made the connection in all these years. Music appreciation is perhaps one of the most subjective and controversial topics. For some people, appreciating music involves understanding the technical nuances and critically evaluating artist’s mastery over the art, and for some of us, it is about simply enjoying the patterns and rhythms. While one might claim that they enjoy all kinds of music, for most of us, only certain kinds of music elicit a deeper appreciation, emotive experience and pleasure. Our music preferences are molded by exposure, cultural diversities and to some extent, mood. Music is extremely varied, and listing the kinds of music could fill pages. Arguing one kind of music is better than other is as like saying one color is better than the other.
So, what biological purpose does music serve? Continue reading