#PromegaWorks

Descriptions of Promega products in action from examples of Promega products in the peer-reviewed literature to technical tips on how to address common laboratory and experimental questions

What I Learned at My First Branch Meeting: Gratitude, Service and Collaboration

Posted on by Promega

Today’s blog post is written by guest blogger Sarah Kolb, Marketing Coordinator for our North America Branch, and new employee at Promega.

As a new member to the North America Marketing team, I was unsure of what to expect going into my first national sales meeting with Promega, but what I took away from this meeting was incredibly eye opening. The North America Branch Sales meeting is an opportunity to get all of the members of the North American branch together to learn about new products, connect with the different strategic business units about product application and network with each other to learn how to better the lives of our customers. The year’s meeting occurred in May in the Ideation room at Promega Headquarters in Madison, Wisconsin.

NA branch meeting

The room itself is not your typical conference space. An antique car resides in the space, and you can find art-work from all over world nestled in corners, and on the walls and shelves. All around the room, collections of unique furniture are arranged to stimulate conversation. Ideation created an atmosphere of creativity, community and collaboration, which contributed to the overall success of the meeting.

I felt excitement Monday morning as the 62 attendees gathered in the space. Everyone greeted each other with big smiles and hugs, asking about families, travels and already discussing business. Continue reading “What I Learned at My First Branch Meeting: Gratitude, Service and Collaboration”

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”

A Better GTPase Assay for Drug Development

Posted on by Sara Klink

Cover of October Issue of Assay and Drug Development Technologies featuring GTPase-Glo™ Assay.The path to drug development is strewn with obstacles: Identifying targets; configuring assays to help identify targets or drugs; uncovering the right compound to affect the selected target without off-target effects and screening multiple compounds to eliminate or identify potential drugs. Without the right tools, compounds or target, identifying potential disease therapies becomes nearly impossible.

When it comes to a drug target for cancer, the Ras protein family is at the top of the list because the proteins are expressed ubiquitiously and found mutated in many types of cancer. Because Ras proteins are involved in transducing signals from the surface of cells, many of the resulting mutations produce an activated Ras, inducing uncontrolled expression of the genes that Ras controls. Ras proteins are small GTPases (20–25kDa) that comprise a larger superfamily of proteins divided into five subfamilies: Ras, Rho, Rab, Arf, and Ran. These proteins control diverse cellular activities, including cellular differentiation, proliferation, cell division, nuclear import and export, and vesicle transport. GTPases are guanosine-nucleotide-binding proteins with affinity for GDP or GTP and are able to hydrolyze GTP. When bound to GTP, GTPases are active (turned on) and interact with downstream proteins in the signaling cascade. When GTPases are bound to GDP, the proteins are inactivated (turned off) and no longer transduce signals. Continue reading “A Better GTPase Assay for Drug Development”

IdeZ Protease: A New Tool for the Characterization of Antibodies

Posted on by Gary Kobs
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Therapeutic monoclonal antibodies are large, complex molecules that undergo numerous post translational modifications (PTMs).  In-depth characterization of antibody PTMs remains a significant hurdle because their large size (~150 kDa) makes mass spectrometry analysis extremely challenging.

IdeS protease specifically cleaves IgGs into Fab and Fc fragments. This enzyme is highly specific and cleaves human IgG specifically at one site in the lower hinge region.  Because of the exquisite specificity of the enzyme, it produces highly homogeneous Fc and Fab fragments which are then readily analyzed using techniques such as mass spectrometry or HPLC.

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One of the drawbacks of IdeS is that it exhibits poor activity against mouse IgGs. IdeZ Protease is an immunoglobulin-degrading enzyme from Streptococcus equi subspecies zooepidemicus. It is an engineered recombinant protease overexpressed in E. coli. Like IdeS Protease, IdeZ Protease specifically cleaves IgG molecules below the hinge region to yield F(ab′)2 and Fc fragments.  Reduction of the digestion products produces three fragments of ~25kDa that are readily analyzed by LC-MS.

One of the key advantages of the IdeZ Protease is that it has significantly improved activity against mouse IgG2a and IgG3 subclasses compared to IdeS Protease. IdeZ Protease does not cleave mouse IgG1 or IgG2b.

Key technical parameters when digesting mouse IgGs utilizing IdeZ are the following:

• Add 1 unit of IdeZ Protease per 1µg of IgG to be digested.
• IdeZ Protease is most active in buffers at or near neutral pH. The recommended digestion buffer is 50mM sodium phosphate, 150mM NaCl (pH 6.6).
• Mouse IgG2a and IgG3 typically require 2–4 hours at 37°C  for complete digestion.
• IdeZ Protease has a histidine tag for easy removal if so desired.

Purify and Conjugate Antibodies in a Single Workflow

Posted on by Gary Kobs

Isoform_Antibodies_LinkedInAntibodies labeled with small molecules such as fluorophore, biotin or drugs play a critical role in various areas of biological research,drug discovery and diagnostics. There are several limitations to current methods for labeling antibodies including the need for purified antibodies at high concentrations and multiple buffer exchange steps.

In a recent publication, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads.

Using a variety of fluorophores the researchers show that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry.

This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Literature Cited

Nidhi, N. et al. (2015) On-bead antibody-small molecule conjugation using high-capacity magnetic bead J. Immunol. Methods  http://dx.doi.org/10.1016/j.jim.2015.08.008

Cell-Free Expression Application: Antibody Screening

Posted on by Gary Kobs

TestPermissions2Ricin, derived from caster seeds, inhibits protein synthesis by binding to ribosomes, resulting in cell death. The protein is composed of two polypeptide chains: Ricin Toxin A chain and Ricin Toxin B chain. Ricin inhibits protein synthesis very quickly, and the cell or tissue damage begins within several hours. However, signs of poisoning often are not noted before significant damage has been done, making treatment difficult. Therapeutics that either block the ribosome binding site or compete with the toxin for binding are highly desired. Both antibodies and competitive ligands inhibited binding of the toxin to cell membranes.

A recent publication by Dong et al. (1), described a study to investigate the therapeutic effect of mAb 4C13, a monoclonal antibody against ricin. One of first experiments performed was to determine the general effect the inhibition of protein synthesis induced by ricin using cell-free expression.

In the study, the authors used T3 Coupled Reticulocyte Lysate Systems from Promega. Both ricin and mAb were diluted with saline. Aliquots of ricin (80 ng/ml) were mixed with an equal volume mAbs (1.6μg/ml) or saline alone and incubated at 4 °C for 1.5 h. A total volume of 4μl of sample was added into the reaction system (i.e, T3 Coupled Reticulocyte and plasmid DNA containing the lucifersase gene downstream of T3 RNA phage promoter). After incubation at 30 °C for 1.5 h, the products were cooled at −20 °C for 10 min. A total of 5μl of each reactive product containing synthesized luciferase was mixed with 50μl luciferase assay reagent pre-equilibrated to room temperature, and the fluorescence absorbance was measured immediately with the micro-ELISA Reader.

Positive results obtained from this preliminary experiment, led to more thorough experiments to determine the dosage effect using in vivo models (i.e., cell lines and mice) to characterize the cytotoxicity and binding activity of mAb 4C13. The mAB 4C13 was shown to be a effective in the mouse model.

Dong, N. et al. (2015) Monoclonal antibody , mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine 33, 3836–42

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

Posted on by Michele Arduengo, PhD
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Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring protein:protein interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal.

Continue reading “If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions”

pH Reactive Dyes for Screening Antibody Internalization

Posted on by Gary Kobs
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Antibody drug conjugates (ADCs) are a new class of therapeutic drugs that uses antibodies to deliver highly toxic drug molecules specifically to the cancer cells. A key requirement for ADCs is the ability of antibody to bind to the cancer cells followed by internalization and subsequent release of drug inside the cells leading to cell apoptosis.

Traditionally, selection of lead antibody candidates for ADCs was done in a sequential workflow where antibodies were first selected based on their affinity followed by characterization involving antibody internalization and drug conjugation. However, there is evidence that high affinity doesn’t always correlate with good internalization and hence there is a need to screen antibodies for internalization properties in addition to their affinities.

Promega has developed a method that allows antibody 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 antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane they are not fluorescent but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dye becomes fluorescent.

Fluorescence signal, for pHAb dyes conjugated using either amine or thiol chemistry, is minimal at pH>7 and increase significantly as the pH drops to pH 5.0, which is a typical pH in cell endosomal compartment. Moreover, pH response of free pHAb dye is similar to that of conjugated dye indicating that conjugation chemistry doesn’t influence the pH response of the dye.

Due to the high signal-to-background ratios of the dyes, plate-based internalization assays can be performed, enabling screening of large libraries of antibodies for their internalization properties, hopefully leading to improved identification of lead candidates for ADC applications.

Avoid False Hits During Compound Screening for Drug Discovery

Posted on by Promega

One goal of drug discovery and research programs is to reduce false hits as early as possible in the process. Follow-up on false hits is costly in terms of time and resources, and the longer the false hits remain in the drug development pipeline, the more costly they are. So methods that can easily reduce the number of false hits during compound screening early in the discovery process are particularly sought after.

Reporter assays have proven to be invaluable tools for elucidating the mechanisms of action of small molecules or other agents on signaling pathways within cells, and the luciferase reporter assay has become a standard research tool in the biological research laboratory.

However, one caveat of using standard luciferase-based reporter assays for larger-scale compound screening efforts is the frequency of false hits that result from direct interaction of compounds with the luciferase reporter. This issue can be mitigated with a “coincidence reporter” system where two independent reporter proteins are produced from a single transcript. In this type of assay, a bicistronic transcript is stoichiometrically translated into two nonhomologous reporters by means of a 2A “ribosomal skipping” sequence. Since it is unlikely that compounds will interact with two distinct types of reporter, “coincident” responses will indicate on-target activity. Such a coincident reporter system provides an important control against costly false hits early in drug discovery research programs.

A paper published online in ACS Chem Biol in February describes the first successful application of the firefly/NanoLuc luciferase coincidence reporter system to identify new pathways that up-regulate PARK2 expression.

Continue reading “Avoid False Hits During Compound Screening for Drug Discovery”

DNA Purification from Plants: Not All Methods are Equal

Posted on by Kelly Grooms

pestle and mortar with leavesIsolating DNA from plant tissues is difficult for many reasons.  Unlike animal cells, plant cells have rigid cell walls, often made of tough fibrous material, and contain proteins and enzymes and other compounds such as polysaccharides and polyphenols that play a role in different cellular processes. These compounds can interfere with DNA isolation as well as downstream applications such as PCR.  For these reasons, DNA isolation methods that are used successfully for other sample types may not work well to isolate DNA from plant material. Continue reading “DNA Purification from Plants: Not All Methods are Equal”