The Making of a Promega Product: Teamwork = Success for the Maxwell RSC® ccfDNA Plasma Kit

Posted on by Nicole Sandler

Ever think about the kinds of challenges R&D scientists run up against in the course of developing a new product? The development of the Maxwell® RSC ccfDNA (circulating cell-free DNA) Plasma Kit is a particularly interesting example. Its path to commercialization was characterized by a number of unexpected technical hurdles, yet each was overcome through creative troubleshooting and aided by valuable collaborations across departments. All had a hand in finally launching the kit last August.

26062525-ccfDNA_Mar25-blog-DNAThe product’s launch was an exciting milestone for Promega as research interest in the role of ccfDNA as biomarkers in human disease continues to grow. Elevated levels of ccfDNA have now been reported in patients with cancer, inflammatory disease, infections and cardiovascular disease. In pregnant women, up to 10% of ccfDNA can be attributed to the fetus, so critical fetal DNA analysis can now be conducted through maternal blood samples. There are many advantages in the ability to isolate and analyze ccfDNA, so the development of a kit with high throughput capability was a priority for the Nucleic Acid Purification R&D team. Continue reading “The Making of a Promega Product: Teamwork = Success for the Maxwell RSC® ccfDNA Plasma Kit”

Inflammasome Research: A Tool to Aid Progress

Posted on by Kari Kenefick

Inflammasomes: A Few Basics

Inflammasomes are protein complexes composed of immune system receptors and sensor molecules. These complexes can respond to both infectious organisms and molecules derived from host proteins. When activated, a series of receptors and molecules signal via either pathogen-associate molecular patterns (PAMPs) induced by microbial pathogens, or danger-associated molecular patterns (DAMPs) induced as a result of endogenous stressors; the common next step in signaling is through pattern recognition receptors (PRR).

Inflammasome diagram.
Innate immune response in inflammation; a basic diagram.

Inflammasome activation is integral to the host immune response in mice and humans (1). The activation results in signaling that activates the caspase-1 scaffold, causing release of immune mediators such as interleukins IL-1β and IL-18. So, whether inflammation is host tissue- or pathogen-induced, inflammasome activation results in a cascade of receptor signaling and mediator release, of which caspase-1 is a critical component.

Continue reading “Inflammasome Research: A Tool to Aid Progress”

Optimizing Antibody Internalization Assays: pHAb Dyes

Posted on by Gary Kobs
22255190_pHabAmine_3D_image_050615-final-large

Promega has recently developed a method that allows antibodies to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When an antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane, the antibody-dye-antigen complex is not fluorescent, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops, and the dye becomes fluorescent.

To demonstrate the broad utility of the pHAb dye for receptor mediated antibody internalization, two therapeutic antibodies, trastuzumab and cetuximab,which bind to HER2 and EGFR respectively, were selected for a case study (1). Both the antibodies, which are known to internalize were labeled with pHAb dyes using amine or thiol chemistry.

Parameters such as the impact of dye–to-antibody ratio on the antigen–antibody binding, change in fluorescence as a function of pH of free dye and labeled dye, and labeled antibody internalization as a function of pHAb conjugated antibody concentration were evaluated.

The results indicate that pHAb dyes are pH sensitive fluorescent dyes that enable the study of receptor-mediated antibody internalization.Internalization assays can be performed in a plate-based homogeneous format and allow endpoint assays as well as real-time monitoring of internalization. They further show that internalization can be monitored even at a very low amount of antibody which is very important during the early monoclonal antibody development phase when the amount of sample is limited and the antibody concentration in the samples is low. a complimentary approach, they  also showed that a secondary antibody labeled with pHAb dye can be used instead of labeling primary antibodies.

Literature cited

Nath, N. et al. (2016) Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye J.  Immunol. Methods epub ahead of print

Helix® Personified

Posted on by Promega

Have you ever thought about life from the point of view of a Helix® On-Site Smart Inventory Unit? We did, and this is what we imagined…

helixtouchsceenisolatedWow! There were a lot of people in and out of here today.

The post doc who never goes anywhere without her earbuds in. I wonder what her research playlist is, she’s always dancing her way in and out of the hall. The new grad student in the virology lab next door. He’s looking a little lost right now.

And of course there’s the PI with the big boots. Never looks where he’s going and constantly almost plowing people down. It’s kind of funny actually, the near misses I’ve seen between him and grad students running out of here with that key reagent they need to finish those last few reaction tubes.

There was a big-name speaker today for the lunch time journal club, so there was a lot of activity on the hall this afternoon. Lots of talking about the presentation. Everyone sounded pretty excited about what they heard.

But now it’s quiet. Most people have gone home for the evening.

Except for the security lighting, the hall is dark.

Oh wait, I hear footsteps heading my way. Apparently, not everyone has stopped work for the day, apparently.

Oh, it’s you. One of my favorite after-hour scientists.

You open the door to the common room, and check the product list on your phone: 100bp DNA ladder … there’s one left. Drawer D.

You pull the pass out of your pocket. I scan it. Click. The door unlocks. You grab your package and let the door close.

I’ll take it from here so that you can get back to work.

Your pass tells me that you’re a post doc in a lab upstairs. After you close the door, I check to see what you took. My scan reads the tag on each reagent. G2101: 100bp DNA ladder. Drawer D.

That was the last one. I update the inventory list. More will arrive in the next resupply shipment.

I send you an email with the details.

Time to check my systems.

Power? Check.
Connection to Promega? Check.
Door closed and locked? Check.
Storage temperature holding steady? Check.
All reagents present? Check.

The lights go dark as I transmit the report back home.

Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts

Posted on by Michele Arduengo, PhD

26911030-Laymans-KSPS-figure-WEB-R4Drug research and development is a complex and expensive process that begins with initial screening steps of candidate chemical compounds, and compounds that appear to have the desired potency against a specific cellular target or pathway are further evaluated. Candidate compounds that fail late in development or during clinical trials because of off-target effects are costly, and can be dangerous. Therefore drug developers not only need to ensure that a candidate compound is effective as a therapy, but also they need to predict any potential undesirable side effects due to off-target activities as early as possible in the drug discovery and development process. Continue reading “Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts”

There and Back Again: Part IV (South Island, New Zealand)

Posted on by Promega

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue is Part IV and the final installment of the adventures of Mica Zaragoza, a senior client rep in Chicago, IL, who used his award to travel to Australia and New Zealand.

26061846-Helix-Travel---Image-1-WEBQueenstown, New Zealand 

Walking out of our room into the open air, Queenstown instantly provided a lasting impression.  Nestled along Lake Wakatipu, watched over by the Remarkable Mountains, the city feels like Aspen on steroids—it’s no wonder there’s such an international pull for young travelers and skiers.

We jumped right into exploring with a gondola ride. This photo is my best attempt at capturing Queenstown from high above. Continue reading “There and Back Again: Part IV (South Island, New Zealand)”

To Meditate Perchance to Dream

Posted on by Kari Kenefick

First the disclosure: this blog is of course about Me.

But it’s also about You. And yours. Because as you know, we’ve become a culture that does not sleep.

Why don’t we sleep? I like to think that it is an evolutionary adaptation; not sleeping, after all, allows us more time for Facebook.

Or Etsy for you makers. Or Amazon for you shoppers. And let’s not forget our middle, high school and college students. Do they even have classrooms anymore, or are lectures all online (on screens)?

One tired pony. By Rachel C from Scotland (Flickr) [CC BY 2.0 , via Wikimedia Commons
One tired pony. By Rachel C from Scotland (Flickr) [CC BY 2.0 , via Wikimedia Commons
Honestly, the evolutionary adaptation idea comes from how we live and work today. And no, this is not another rant/lecture on the color of light emitted by whatever non-cathode ray tubes are in our phones or tablet-like devices.

It’s just that just working in our very busy online/wired world, jumping from web page to project management software, to big-screens in meetings has us adapted to being  on: capital “O” capital “N”.

This multi-multitasking has grown (for me) a new type of neurons that are not happy unless they are gleaning new information from a screen, all the time. And these neurons don’t stop working when the screen is gone; no, they continue seeking and trying to process. For me, if there’s no screen to look at, the neurons ping-pong around behind my eyeballs, looking and searching, as if to say, “Input missing! Input missing!”

The result can be hours in bed sans sleep; it seems the racket these neurons make keeps all the other neurons up. Continue reading “To Meditate Perchance to Dream”

Congenital Cytomegalovirus: The Most Common Congenital Infection That You Have Never Heard Of

Posted on by Kelly Grooms
infant

Down Syndrome. Fetal alcohol syndrome. Spina bifida. Most people have heard of these well know congenital conditions, and know at least in a general sense that they have profound and lasting effects on the children born with them as well as their families. Unfortunately, people are much less aware of a congenital infection that is more common that any of these and affects more infants than all three of the conditions listed above. In fact, this congenital infection causes more cases of congenital disease than all of the 29 conditions currently screened for in most American states combined (1; 2), and yet it is not widely known by the general public. Cytomegalovirus, or CMV, is the most common congenital viral infection in developed countries (3; 4), and the leading cause of congenital sensorineural hearing loss and psychomotor retardation (1).

Continue reading “Congenital Cytomegalovirus: The Most Common Congenital Infection That You Have Never Heard Of”

T-Vector Cloning: Answers to Frequently Asked Questions

Posted on by Radhika Ganeshan
Blue/White colony screening helps you pick only the colonies that have your insert.
Blue/White colony screening helps you pick only the colonies that have your insert.

Q: Can PCR products generated with GoTaq® DNA Polymerase be used to for T- vector cloning?

A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq®DNA Polymerase lacks 3’ →5’ exonuclease activity (proof reading) and also displays non-template–dependent terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning.

We have successfully cloned PCR products generated using GoTaq® and GoTaq® Flexi DNA Polymerases into the pGEM®-T (Cat.# A3600) and pGEM®-T Easy (Cat.# A1360) Vectors.

Q: Can GoTaq® Long PCR Master Mix be used for T-Vector Cloning?

A: Yes it can. GoTaq® Long PCR Master Mix utilizes recombinant Taq DNA polymerase as well as a small amount of a recombinant proofreading DNA polymerase. This 3´→5´ exonuclease activity (proof reading) enables amplification of long targets. Despite the presence of a small amount of 3´→5´ exonuclease activity, the GoTaq® Long PCR Master Mix generates PCR products that can be successfully ligated into the pGEM®-T Easy Vector System.

We have demonstrated that GoTaq® Long PCR Master Mix successfully generated DNA fragments that could be ligated into pGEM®-T Easy Vector System without an A-tailing procedure, and with ligation efficiencies similar to those observed with the GoTaq® Green Master Mix.

For details refer to Truman, A., Hook, B. and Wieczorek, D. Using GoTaq® Long PCR Master Mix for T-Vector Cloning.

Tip: For cloning blunt-ended PCR fragments into T-vectors, use the A-tailing protocol discussed in the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

Q: How do I prepare PCR products for ligation? What products can be used to purify the DNA?

Continue reading “T-Vector Cloning: Answers to Frequently Asked Questions”

What’s for Dinner? Mystery Meat Served at the 47th Explorer’s Club Annual Dinner Finally Identified

Posted on by Michele Arduengo, PhD
Could this be the 1951 mystery meat?
Could this be the 1951 mystery meat?

I remember one particular encounter with “mystery meat” when I was in college. I was walking along the serving line at the dining hall, and when I came to the entrée, I asked the server, “What is it?”

She replied quite succinctly, “Don’t know. Got beef in it.” I passed on the entrée that night, settling for salad and bread.

I would probably not have be a good candidate for membership in the Explorers Club.

The Explorers Club, founded in New York City in 1904, is a professional society that champions the cause of field research (1). The member list is impressive, including Teddy Roosevelt, the American President responsible for setting aside many of the most treasured public lands in the United States so that explorers have fields for research and wild places for adventures, Neil Armstrong, the first man to set foot on the moon, and Don Walsh and Jacques Piccard, the two men who descended into the Mariana’s trench to explore the deepest part of the ocean, among others.

In addition to a membership list that reads like a who’s who of science and exploration, The Explorers Club also has an annual dinner that for many years has popularized a menu of “exotic” foods (at least exotic foods from the point of view of the typical Midwest United States pallet). One of the club’s most celebrated dinners took place on January 13, 1951.

Continue reading “What’s for Dinner? Mystery Meat Served at the 47th Explorer’s Club Annual Dinner Finally Identified”