Many studies, from reporter assays to protein localization to BRET and FRET, require successful transfection first. Yet, transfection can be tricky and difficult. There are many considerations when planning transfection of your cells including reagent selection, stable or transient experiment, type of molecule and endpoint assay used. Here we discuss these considerations to help you plan a successful transfection scheme for your experimental system. Continue reading “General Considerations for Transfection”
How Do You Defrost the Lab Freezer?
When I was in the lab, we usually started with an elaborate system of borrowed hairdryers and old chemistry ring stands. What is your preferred method of attacking a frost-full freezer?
Differentiating but not Mature Adipocytes Provide a Defense Against S. aureus Infection
A basic tenet of immunology is that antibodies produced by B cells are very important and specific immunoprotective agents, released in response to infection.
However, antibodies do not supply immediate protection. The invading organism needs to get into the host, meet up with T cells and then B cells, in order for antibody production to occur. If the host has seen this particular pathogen previously, the antibody response occurs somewhat more quickly, but we’re still talking about days. If the invading organism is a bacterium, it can multiply and double in numbers in just hours. Thus an infection could potentially gain a foothold in a body prior to an antibody response.
Fortunately we have a more rapid, first line of defense to invading pathogens, a cellular response. In the case of a puncture or skin wound, epithelial cells, mast cells and leukocytes are activated quickly in response to pathogens. Neutrophils and monocytes also aid the cellular response.
Now a recently published report demonstrates that fat cells also play a part in the cellular response to invading bacteria. R. Gallo et al. published a study on Jan. 2 in Science, providing more in depth information on the role of adipocytes in the host response to the bacterium Staphylococcus aureus (S. aureus). Continue reading “Differentiating but not Mature Adipocytes Provide a Defense Against S. aureus Infection”
Improved Characterization and Quantification of Complex Cell Surface N-Glycans
N-Glycosylation is a common protein post-translational modification occurring on asparagine residues of the consensus sequence asparagine-X-serine/threonine, where X may be any amino acid except proline. Protein N-glycosylation takes place in the endoplasmic reticulum (ER) as well as in the Golgi apparatus.
Approximately half of all proteins typically expressed in a cell undergo this modification, which entails the covalent addition of sugar moieties to specific amino acids. There are many potential functions of glycosylation. For instance, physical properties include: folding, trafficking, packing, stabilization and protease protection. N-glycans present at the cell surface are directly involved in cell−cell or cell−protein interactions that trigger various biological responses.
The standard method used to profile the N-glycosylation pattern of cells is glycoprotein isolation followed by denaturation and/or tryptic digestion of the glycoproteins and an enzymatic release of the N-glycans using PNGase F followed by analysis mass spec. This method has been reported to yield high levels of high-mannose N-glycans that stem from both membrane proteins as well as proteins from the ER.(1,2)
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For those researchers interested in characterizing only cell surface glycans (i.e., complex N-glycans) a recent reference has developed a model system using HEK-292 cells that demonstrates a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells (3). The method involves standard centrifugation followed by enzymatic release of cell surface N-glycans. When compared to the standard methods the detection and quantification of complex-type N-glycans by increased their relative amount from 14 to 85%.
- North, S. J. et al. (2012) Glycomic analysis of human mast cells, eosinophils and basophils. Glycobiology. 2012, 22, 12–22.
- Reinke, S. O. et al. (2011) Analysis of cell surface N-glycosylation of the human embryonic
kidney 293T cell line. J. Carbohydr. Chem. 30, 218–232. - Hamouda, H. et al. (2014) Rapid Analysis of Cell Surface N‑Glycosylation from Living Cells Using Mass Spectrometry. J of Proteome Res. 13, 6144–51.
Monday SMILE
Monday lab meeting? Journal club? Long weekend in the lab?
Enjoy this classic from the Promega Cartoon Lab.
How MicroRNAs Have a Big Effect on Genetic Regulation
Some of us scientists who have been around for a while still think about RNA molecules falling into three categories: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). However, I have revised my outdated RNA classification scheme as scientists discover exciting new classes of RNAs that do some fairly amazing things. For example, in the early 1980s, Thomas Cech discovered ribozymes, RNAs that have catalytic functions (1), and in the early 1990s, researchers began to take interest in short noncoding RNAs that act as a genetic regulators, the first of which was discovered in C. elegans (2). RNA is no longer simply a biological middleman between DNA and protein. These ephemeral nucleic acid molecules play a much bigger role of cellular physiology and gene regulation than we had previously ascribed to RNA.
Continue reading “How MicroRNAs Have a Big Effect on Genetic Regulation”Reflecting on the Future: Hands-On, Person-to-Person Educational Experiences
The start of a new year is always a good time for reflection. For those of us at the BioPharmaceutical Technology Center Institute (BTC Institute), this means looking at the programs we offer and considering ones we might like to develop.
In this process, we find ourselves continuing to feel certain that the hands-on, lab-based opportunities we provide add something meaningful to the education of those we serve, from middle school students and their teachers to graduate students to scientists in academia and industry. The value of learning concepts and techniques in a well-equipped setting, working with teachers and volunteers who are dedicated scientists, is significant.
In addition to gaining an understanding of the basics of molecular biology so key to biotechnology, these programs are also designed to support the development of critical thinking skills so necessary to scientific literacy.
We think this is also the case for our scientific symposia (Wisconsin Stem Cell Symposium; Wisconsin Human Proteomics Symposium) and our International Forum on Consciousness. These events enable attendees to interact with speakers and other participants in person – in an environment designed to encourage the exchange of information, ideas and perspectives. Continue reading “Reflecting on the Future: Hands-On, Person-to-Person Educational Experiences”
Happy Holidays from Promega Connections
Your Promega Connections Bloggers will be taking a break from December 23, 2014–January 5, 2015, to retool and gather up our thoughts so that we can cook up New Year of intriguing science posts. You can look forward to posts about bioluminescence, cytotoxicity and cell culture, kinases and cellular signaling networks, epigenetics, and the new twists and turns in biotechnology that are making their way into our labs and our lives. We will continue our tradition of posts to guide the job seekers and those of you in career transitions, and of course we will provide lots of technical tips and tricks for your work at the bench.
If you have any burning technical questions or a science or technology topic on which you would like to see a post, let us know in the comment section below.
Be sure you are subscribed to the RSS feed so that you can join us on our blogging journey in 2015.
Happy New Year!
NO! I CAN do that.
Today’s blog is written by guest blogger Jessica Laux, a production scientist at Promega Corporation. Jessica spends most of her time in clean rooms. She graduated from the University of Wisconsin-Madison with a B.S. in Natural Science-Animal Sciences.
I was always a very stubborn, defiant child. This is evidenced by the fact that my very first word was “NO!”, which I screamed at the top of my lungs after I had been scolded for pulling all the pots and pans out of the kitchen cupboards. Years later, I still scream “NO!” at times, though I’ve refrained from making a mess of the kitchen lately. That same defiant spirit contributed a great deal to my chosen career.
At a ripe age of ten, I determined I was destined to become a great doctor. My preparation for this career involved writing morbid stories where I brought the dead back to life, as well as poring over the pages of a medical diagnostic book I had claimed as mine. I was not deterred by my inability to understand the big words. I was still able to draw the detailed human anatomy and skeletons with an impressive precision. A couple years later, an adult whom I trusted told me that science and medicine were fields for men only. This same person encouraged me to pursue my artistic talents instead. Continue reading “NO! I CAN do that.”
Tell Me Your Story
Next time you have a job interview, try not to think of it as a question and answer session, but instead think of it as an opportunity to tell your story. Boring questions tend to lead to boring answers, so that’s what recruiters and hiring managers often get. Before reciting a canned answer to the question “How would you describe your leadership style?” or “What is your greatest strength?”, take a step back and come up with a story to explain your answer. You’ll come across as more charming, a great communicator and the interviewer will get a chance to know you better. Continue reading “Tell Me Your Story”
