Practical Tips for HEK293 Cell Culture When Using cAMP-Glo™ Assay

HEK293 cells stably expressing HaloTag®-ECS (ExtraCellular Surface; comprised of a signal sequence and single transmembrane domain of β1-integrin) fusion protein labeled with HaloTag® Alexa Fluor® 488 Ligand and then imaged.
HEK293 cells stably expressing HaloTag®-ECS (ExtraCellular Surface; comprised of a
signal sequence and single transmembrane domain of β1-integrin) fusion protein labeled
with HaloTag® Alexa Fluor® 488 Ligand and then imaged.
G Protein Coupled Receptors represent one of the largest classes of cell surface receptors and one of the most important classes for drug targets. Fifty of the top 200 drugs target GPCRs. GPCRs respond to various stimuli like light, odors, hormones, neurotransmitters and others. They cover virtually all therapeutic areas. When a particular GPCR is implicated in a disease, researchers screen the GPCR and its signaling pathways, the hope being that promising therapeutic targets might be identified. Major G-protein families signal via secondary messengers like cAMP, which in turn activate a range of effector systems to change cell behavior and/or gene transcription. There are various approaches and methods to study GPCRs and measure the increase or decrease of intracellular cAMP. However, the fastest and the most sensitive among all methods is a plate based cAMP-Glo™ Assay. Continue reading “Practical Tips for HEK293 Cell Culture When Using cAMP-Glo™ Assay”

Breast Cancer(s) Heterogeneity and Personalized Treatments

Image used with permission of Dr. Hongdi Liu.
Image used with permission of Dr. Hongdi Liu.
Recently Promega hosted a special guest from Duke University—Dr. Neil. L. Spector, one of leading scientists in the field of breast cancer research. In a simple way  Dr. Spector presented advances in this field of cancer research and informed us of new treatments that have the potential to improve patient lives. Continue reading “Breast Cancer(s) Heterogeneity and Personalized Treatments”

Optimizing a DNA Methylation Analysis Workflow

methyledge seminarWhen Aristotle compared epigenetics to a net (1), he could not have predicted how right he was.  Recent research has revealed that mechanisms underlying epigenetic effects are numerous and interdependent as are the knots in a net. Each epigenetic mechanism has its players: enzymes, functional groups, substrates etc.  The most important aspect of an epigenetic trait is its reversibility. Methylation of DNA was the first epigenetic modification to be discovered, and 5-cytosine methylation was the first to be linked with gene expression status. Currently, the most popular method for measuring  CpG island methylation status is a bisulfite treatment of DNA followed by PCR or sequencing.

In this week’s webinar, Promega R&D scientist, Karen Reece focused on a workflow from DNA purification to analysis. She described the best methods for DNA isolation, quantification, bisulfite conversion, PCR and sequencing. Continue reading “Optimizing a DNA Methylation Analysis Workflow”

ProK: An Old ‘Pro’ That is Still In The Game

Proteinase K Ribbon Structure ImageSource=RCSB PDB; StructureID=4b5l; DOI=http://dx.doi.org/10.2210/pdb4b5l/pdb;
Proteinase K Ribbon Structure ImageSource=RCSB PDB; StructureID=4b5l; DOI=http://dx.doi.org/10.2210/pdb4b5l/pdb;
If you enter any molecular lab asking to borrow some Proteinase K, lab members are likely to answer: “I know we have it. Let me see where it is”. Sometimes the enzyme will be found to have expired. The lab may also have struggled with power outages or freezer malfunctions in the past. But the lab still decides to keep the enzyme. One may rightly ask – why do labs hang on to Proteinase K even when it has been stored under sub-standard conditions? Continue reading “ProK: An Old ‘Pro’ That is Still In The Game”

Decorating Histones and Their Tails

There are at least two tail stories associated with big scientific discoveries. One is Darwin’s story about the tail loss during human evolution process. The other story is associated with discovery of benzene ring structure. In his creative dream Kekule saw the snake (the linear carbon chain) eating its own tail. Even better representation of benzene structure is the comic image of six monkeys holding each other hands and tails. Nowadays, the most popular scientific story in the field of epigenetics is the story of histones and their tails. This time instead of monkey or snake, an elephant is the animal whose characteristics allegorically represent epigenetics.

elephants_web

We all know how the histone octamer wrapped with DNA represents a nucleosome – the first unit of chromatin formation. Histones, which are basic due to numerous arginines and lysines, easily attract negatively charged DNA and in that way facilitate formation of nucleosome. The nature of two materials is important but not sufficient for such complex biological function like efficient packaging of DNA and regulation of gene expression. For that reason both the DNA and histones are decorated by numerous chemical groups.

Post-translational modifications (PTMs) of histones and histone variants themselves can cause alternation of net charge, changes histone dynamics and interaction with other chromatin proteins. The extreme complexity of interactions that can be achieved by histone modifications inspired Jenuwein and Allis to launch an idea of “histone or epigenetic code”. Core histones consist of a N- terminal tail, the globular portion and a C terminus. PMTs were discovered first on the N-terminal tail of core histones. However, the logical question was: Are only the tails decorated or are there more?” Continue reading “Decorating Histones and Their Tails”

How to Choose a Good Reference Gene?

A Researcher’s work is never easy but it is even harder when relative data are to be interpreted. This is especially true for Real-Time PCR. It is one of the most accurate ways to evaluate gene expression. However, despite it being such a powerful technique, it still carries many pitfalls which can lead a scientist to the wrong conclusion. Often a new user does not have thorough sample/RNA preparation, equipment or knowledge. So what are the considerations and aspects that the researcher should pay attention to? Continue reading “How to Choose a Good Reference Gene?”

Imperfect Crystal – Inclusion Body

Protein Crystals. Image courtesy of NASA.
Formation of inclusion bodies is one of the most common complications in heterologous protein expression (1). Despite this complication, the E. coli expression system is still highly used for eukaryote protein expression. Is this practice based on knowledge or historic consequence? Continue reading “Imperfect Crystal – Inclusion Body”

Nanoparticles – Workhorses That Bring Tremendous Benefit

Tiny particles found in clothing, cosmetics, food, electronics or furniture enter our bodies and behave in unexpected (sometimes unwanted) ways. However, in the realm of medicine another type of particle called the nanoparticle can bring untold potential. We can load them with drugs, for example, and deliver them precisely to a diseased organ or cell. Mark Davis from the California Institute of Technology has created nanoparticles that deliver siRNA specifically into melanomas. Davis and his colleagues have not shied away from making bold claims about the therapeutic potential of their work.  They write:

“When taken together, the data presented here provide the first, to our knowledge, mechanistic evidence of RNAi in a human from an administered siRNA. Moreover, these data demonstrate the first example of dose-dependent accumulation of targeted nanoparticles in human tumours. ….These data demonstrate that RNAi can occur in a human from a systemically delivered siRNA, and that siRNA can be used as a gene-specific therapeutic.” Davis et al. 2010. Continue reading “Nanoparticles – Workhorses That Bring Tremendous Benefit”