Author: Promega

Which DNA Do I Use? How to Choose Your Control and Other DNA Samples

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DNA double helix molecules and chromosomes.

Today’s Promega Connections blog is written by guest blogger Joliene Lindholm, Promega Technical Services Scientist.

In Promega Technical Services, we are frequently asked questions about choosing among our Human Genomic DNA products. Promega offers DNA that can serve as sources of normal human gene sequences or positive controls where genotype is not critical, and controls for use in genotyping applications like STR analysis. For mouse researchers, we also offer Mouse Genomic DNA. Continue reading “Which DNA Do I Use? How to Choose Your Control and Other DNA Samples”

National Optimism Month—Make it a Year

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People working togetherToday’s Promega Connections blog is written by guest blogger Tori Sheldon, North America Marketing and Events Coordinator.

It is crazy to think how quickly the months fly by. It feels like yesterday I was watching the ball drop as 2017 turned to 2018. Now it is almost March, when Wisconsin starts to emerge from the cold winter. March also happens to be National Optimism Month.

As I think about optimism, I am reminded of one of the core values that guide interpersonal relationships at Promega: “look for the good, with discernment”.  The spirit of this value is to remember that deep down everyone is trying to come from a positive place and that even though we may not always agree with each other it is an opportunity for further discussion and collaboration.

This value is a part of the Emotional & Social Intelligence (ESI) program at Promega. Continue reading “National Optimism Month—Make it a Year”

Adventure in Belize

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Today’s blog post is written by guest blogger, Josh Agate, Manager, Global CRM.

Picture from airplane approaching Ambergis Caye
Approaching Ambergis Caye.

Adventure is relative. Most people are looking for new adventures in life, and those can range from planning where to go on vacation to starting a new job. What each person looks for in an adventure and the level of thrill they seek is different. When I learned that Promega had awarded me a trip to a destination of my choice with my family for my job performance, I was excited to plan this new adventure with my wife and two daughters (ages 4 and 6). We decided on a trip to Belize.

The trip required two commercial flights, followed by a puddle jumper flight (with hand-written boarding passes), and a 30 minute boat ride before we arrived at our hotel on the island of Ambergris Caye.  This island, off the northern coast of Belize, would provide the backdrop for our family’s greatest adventure to date. The trip to get to the island wasn’t tedious travel for them; it was a wild ride that included a plane that held 12 people, flying over crystal clear waters and a boat trip, where our hair flew wildly as we were sprayed with ocean mist. Continue reading “Adventure in Belize”

Mass Spec Analysis of PTMs Using Minimal Sample Material

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DNA is organized by protein:DNA complexes called nucleosomes in eukaryotes. Nucleosomes are composed of 147 base pairs of DNA wrapped around a histone octamer containing two copies of each core histone protein. Histone proteins play significant roles in many nuclear processes including transcription, DNA damage repair and heterochromatin formation. Histone proteins are extensively and dynamically post-translationally modified, and these post-translational modifications (PTMs) are thought to comprise a specific combinatorial PTM profile of a histone that dictates its specific function.  Abnormal regulations of PTM may lead to developmental disorders and disease development such as cancer.

Continue reading “Mass Spec Analysis of PTMs Using Minimal Sample Material”

Promega Travel Award Blog: An Excursion to Croatia

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Dubrovnik, the Pearl of the Adriatic
Dubrovnik, the Pearl of the Adriatic

We invite you to travel with Bettina Bazzini-Lapin, Scientific Client Specialist, who was awarded a Promega Travel Award for sales performance and used her award to travel to Croatia and Italy. In this blog, she describes her travels.

Croatia is an Eastern European country that sits on the Adriatic Sea directly across from Italy. It has more than one thousand islands, and a third of the country is covered by forest. It is known for its beautiful Dalmatian coast line. One of the main sites for travelers to visit is the coastal city of Dubrovnik, known as the Pearl of the Adriatic. This is where my adventure began. Continue reading “Promega Travel Award Blog: An Excursion to Croatia”

Optimized Detection of EPO-Fc in Human Biological Fluids

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Recombinant erythropoietin (rhEPO) is often used as “doping agent” by athletes in endurance sports to increase blood oxygen capacity. Some strategies improve the pharmacological properties of erythropoietin (EPO) through the genetic and chemical modification of the native EPO protein. The EPO-Fcs are fusion proteins composed of monomeric or dimeric recombinant EPO and the dimeric Fc region of human IgG molecules. The Fc region includes the hinge region and the CH2 and CH3 domains. Recombinant human EPOs (rhEPO) fused to the IgG Fc domain demonstrate a prolonged half-life and enhanced erythropoietic activity in vivo compared with native or rhEPO.

Drug-testing agencies will need to obtain primary structure information and develop a reliable analytical method for the determination of EPO-Fc abuse in sport. The possibility of EPO-Fc detection using nanohigh-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) was already demonstrated (1). However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In a recent publication, researchers describe the effort to identify peptides covering unknown fusion breakpoints (later referred to as “spacer” peptides; 2). The identification of “spacer” peptides will allow the confirmation of the presence of exogenous EPO-Fc in human biological fluids.

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Explore our portfolio of proteases today.

A bottom-up approach and the intact molecular weight measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified.

Results indicated that “spacer peptides” could be used in the determination of EPO-Fc fusion proteins in biological samples using common LC−tandem MS methods.

References

  1. Reichel, C. et al. (2012) Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.
    Drug Test. Anal. 4, 818−29.
  2. Mesonzhnik, N. et al. (2017) Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography−Mass Spectrometry.
    J. of Proteome Res. 17, 689-97.

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

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NanoLuc dual reporters
Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading “Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit”

Determination of Antibody Mechanism of Action Using IdeS Protease

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Monoclonal antibodies (mAbs) have been widely used to eliminate undesired cells via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and programmed cell death (PCD). Unlike the Fc-dependent mechanism of ADCC and CDC, certain antibody–antigen interactions can evoke direct PCD via apoptosis or oncosis. Previously, researchers have reported the specific killing of undifferentiated human embryonic stem cells (hESC) by mAb84 (IgM) via oncosis (1)

In a recent publication (2), a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC). One of the many experimental tools used to characterize the mechanism of oncosis was the fragmention of the A1 antibody with IdeS and papain.

Learn more about IdeS and IdeZ Protease available from Promega.

Papain digestion of IgG produces Fab fragments in the presence of reducing agent. F(ab)2 fragments of A1 were produced using IdeS Protease.

The results indicate that both Fab_A1 and F(ab)2_A1 bind to hESC but only F(ab)2_A1 retained hESC killing. Hence bivalency, but not Fc-domain, is essential for A1 killing on hESC.

  1. Choo, A.B. et al. (2008) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells  26, 1454.
  2. Zheng, J.Y. et al. (2017) Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis. Cell Death and Differentiation 24, 546–58.

Are you looking for proteases to use in your research?
Explore our portfolio of proteases today.

Honoring Caregivers

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This blog post is contributed by guest blogger Diana Clark, Benefits Manager, Promega Corporation

November is National Family Caregivers Month, first proclaimed by President Clinton in 1997, the proclamation has been renewed by every U.S. President since. When President Obama proclaimed this designation in 2012, he commented, “The unselfish devotion of family caregivers affirms the importance of respecting the dignity of life in all stages and underscores the importance of the family unit.”

Hearing these words, I felt even prouder to be a part of the Promega family. You see, we are already in the process of rolling out Caregiver Leave for 2018. Caregiver Leave will provide Promega employees with an additional three weeks of paid time off annually to care for a sick parent, spouse or child, or to welcome a new child into their family via birth, adoption or foster placement. Continue reading “Honoring Caregivers”

Analysis of a biosimilar mAb using Mass Spectrometry

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Several pharmaceutical companies have biosimilar versions of therapeutic mAbs in development. Biosimilars can promise significant cost savings for patients, but the unavoidable differences
between the original and thencopycat biologic raise questions regarding product interchangeability. Both innovator mAbs and biosimilars are heterogeneous populations of variants characterized by differences in glycosylation,oxidation, deamidation, glycation, and aggregation state. Their heterogeneity could potentially affect target protein binding through the F´ab domain, receptor binding through the Fc domain, and protein aggregation.

As more biosimilar mAbs gain regulatory approval, having clear framework for a rapid characterization of innovator and biosimilar products to identify clinically relevant differences is important. A recent reference (1) applied a comprehensive mass spectrometry (MS)-based strategy using bottom-up, middle-down, and intact strategies. These data were then integrated with ion mobility mass spectrometry (IM-MS) and collision-induced unfolding (CIU) analyses, as well as data from select biophysical techniques and receptor binding assays to comprehensively evaluate biosimilarity between Remicade and Remsima.

The authors observed that the levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. they found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact.  Importantly, they identified more than 25 glycoforms for each product and observed glycoform population differences.

Overall the use of mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. They demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

1. Pisupati, K. et. al. (2017) A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Anal. Chem 89, 38–46.