Author: Promega

Converting RPM to g Force (RCF) and Vice Versa

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Rotational Radius
Rotational radius of centrifuge for converting RPM to g force (RCF).

g Force or Relative Centrifugal Force (RCF) is the amount of acceleration to be applied to the sample. It depends on the revolutions per minute (RPM) and radius of the rotor, and is relative to the force of Earth’s gravity.

A good, precise protocol for centrifugation instructs you to use the g force rather than RPMs because the rotor size might differ, and g force will be different while the revolutions per minute stay the same. Unfortunately, many protocols are written in hurry and instructions are given in RPMs. Therefore, you have to convert g force (RCF) into revolutions per minute (rpms) and vice versa.

Modern centrifuges have an automatic converter but older ones do not. There is a simple formula to calculate this, but it takes some time to do the calculation. Meanwhile, your cells might die or the biochemical reaction goes on for three times longer than it should.

There are several ways to make conversion:

Continue reading “Converting RPM to g Force (RCF) and Vice Versa”

Characterizing Compound Binding in Cell-Free Systems

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Dioxins (e.g., 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD) and related compounds (DRCs) are persistent environmental pollutants that gradually accumulate through the food chain, mainly in the fatty tissues of animals. Dioxins are highly toxic and can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This broad range of toxic and biological effects of DRCs is mostly mediated by the aryl hydrocarbon receptor (AHR).

In animal cells, DRCs bind to AHR in the cytoplasm and then translocate into the nucleus, where they affect the transcription of multiple target genes, including xenobiotic-metabolizing enzymes, such as CYP1A isozymes. AHR is also involved in immune system maintenance, protein degradation and cell proliferation.

The jungle crow (Corvus macrorhynchos) has been considered a suitable indicator for monitoring environmental chemicals such as DRCs. While mammals only have one AHR form, avian species have multiple AHR isoforms such as AHR1 and AHR2. To unveil the functional diversity of multiple avian AHR isoforms in terms of their contribution to responses to DRCs a recent study by Kim et al. investigated the molecular and functional characteristics of jungle crow AHR isoforms, cAHR1 and jcAHR2 (1).

cAHR1 and jcAHR2 proteins were synthesized using AHR proteins were synthesized using the TnT Quick-Coupled Reticulocyte Lysate System  to examine whether these jcAHRs have the potential to bind to TCDD. TCDD-binding affinity of the in vitro-expressed jcAHR protein was analyzed using the velocity sedimentation assay with a sucrose gradient.

The results demonstrate that both jcAHR1and jcAHR2 are capable of binding to TCDD.

Reference
Kim, E-Y (2019) The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos). Ecotoxicology and Environmental Safety 171. 99–111

From BTCI to Africa and Back Again: One Student’s Journey in Science Education

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Today’s blog is brought to us by and alumus of Dane County Youth Apprenticeship Program, Aidan Holmes.

In this blog I have the opportunity to write about how my experiences at the BTC Institute as a high school student were instrumental in leading me to my passion for science education, my Peace Corps experience, and my current role as a biotechnology instructor for the very same institute.

I became familiar with the BTC Institute as a student at Marshall High School when our biology teacher organized a biotechnology field trip for us. I loved learning about DNA and biotechnology since 7th grade so attending a field trip like this was an incredible opportunity to engage in hands-on biotechnology. When I learned about the Youth Apprenticeship Program in Biotechnology I knew I had to apply and enrolled during my senior year of high school. Through the program I took a weekly class at the BTC Institute and I worked as a student researcher in a biochemistry lab at UW-Madison. I enrolled for classes at UW-Madison the following year and pursued an undergraduate degree in genetics and a certificate in education and educational services. Continue reading “From BTCI to Africa and Back Again: One Student’s Journey in Science Education”

Mutation Analysis Using HaloTag Fusion Proteins

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In a recent reference, Kinoshita and colleagues characterized the phosphorylation dynamics of MEK1 in human cells by using the phosphate affinity electrophoresis technique, Phos-tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Phos-tag SDS-PAGE; 1). They found that multiple variants of MEK1 with diferent phosphorylation states are constitutively present in typical human cells.

To investigate the relationships between kinase activity and drug efficacy researchers from the same laboratory group conducted phosphorylation profling of various MEK1 mutants by using Phos-tag SDS- PAGE (2).

They introduced mutations in of the MEK-1 coding gene that are associated with spontaneous melanoma, lung cancer, gastric cancer, colon cancer and ovarian cancer were introduced into Flexi HaloTag clone pFN21AE0668, which is suitable for expression of N-terminal HaloTag-fused MEK1 in mammalian cells. Continue reading “Mutation Analysis Using HaloTag Fusion Proteins”

Executing a NanoBRET™ Experiment: From Start to Data

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This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”

Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.

In a few words, can you explain NanoBRET?
“NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”

Optimizing Pressure Cycling Sample Preparation for Bottom-Up Proteomics

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Large-scale analyses of the proteome have revealed proteomic changes in response to disease, and these changes hold great promise for diagnostics and treatment of complex diseases if proteomic analysis can be brought into the clinical laboratory. Successful and reliable large-scale proteomics requires sample preparation workflows that are reproducible, reliable and show little variability. To bring proteomics into the clinical laboratory, standardized procedures and workflows for sample prep and analysis are required to generate valid, actionable results on a time scale useful for the clinic.

The two most common sample types analyzed for clinical proteomics are body fluids and tissue biopsies. To process these kinds of samples, there are two initial steps: tissue solubilization, followed by proteolytic digestion. Solubilization of solid tissues is the most labor-intensive and produces the most variable results.

The introduction of pressure cycling technology (PCT) using Barocycler instrumentation has greatly improved both tissue solubilization and digestion consistency. The PCT-based sample preparation protocols generally utilize urea as a lysis buffer for protein denaturing and solubilization. Urea has several drawbacks including inhibiting trypsin activity and introducing unwanted modifications like carbamylation.

Lucas and colleagues analyzed whether replacing urea with SDC would produce similar tissue digestion profiles and improve the PCT method.

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SDC allowed the use of higher temperatures compared to urea, and hence the first step (lysis, reduction, and alkylation) was performed at 56 °C. The second digestion step in the Barocycler was optimized, and the third step was eliminated. To further reduce digestion time, they capitalized on Rapid Trypsin/Lys-C.  Rapid Trypsin/Lys-C maintains robust activity at 70 °C, and allowed Barocycler digestion to be performed in a single step, completing digestion in 30 cycles (approximately 30 min) rather than 105 minutes, streamlining the protocol.

The data presented an improved conventional tissue PCT approach in a Barocycler by replacing urea and proteolytic enzymes with SDC, N-propanol, and modified commercially available enzymes that have higher optimum temperatures.

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Paper Referenced

Lucas, N. et al. (2019) Accelerated Barocycler Lysis and Extraction Sample Preparation for Clinical Proteomics by Mass Spectrometry. J of Proteome Res 18, 399–405.

Related Posts

Oh, the Places You’ll Go, Thanks to Science!

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Today’s guest blog is written by Aparna Shah, a Post-Doc at Johns Hopkins University. Aparna visited the Promega campus in Madison, Wisconsin on January 18, 2019 and offered to share her experiences.

Aparna poses with her artwork at the Promega Employee Art Showcase

I don’t recall ever having won a contest before, let alone the grand prize! In fact, I did a double take when I first read the email informing me that my SciArt submission had been selected as a winning entry for the Promega Art Contest for Creative Scientists. What did I win, you ask? A free trip to Madison, WI to meet with the team behind the contest and explore Promega’s headquarters!

I first heard about the contest on the HelloPhD podcast and considered participating primarily to support the SciArt movement. A couple of days later, I came across a perfectly-timed tweet about the contest that nudged me out of procrastination mode and reminded me to follow through with it. I’m going to take a second here to pitch both HelloPhD and Twitter to you. Regardless of whether you’re an undergraduate student interning in a science lab or a senior postdoc, the HelloPhD podcast is incredible at calming you down while you’re on the roller coaster ride called academia. As for Twitter, I can think of several pros for using it. But in the context of this post, it is one of the best resources for discovering opportunities that match your interests.

Continue reading “Oh, the Places You’ll Go, Thanks to Science!”

Deep in the Jungle Something Is Happening: DNA Sequencing

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This blog was written by guest blogger and 2018 Promega Social Media Intern Logan Godfrey.

Only 30 years ago, the polymerase chain reaction (PCR) was used for the first time, allowing the exponential amplification of a specific DNA segment. A small amount of DNA could now be replicated until there was enough of it to study accurately, even allowing sequencing of the amplified DNA. This was a massive breakthrough that produced immediate effects in the fields of forensics and life science research. Since these technologies were first introduced however, the molecular biology research laboratory has been the sole domain of PCR and DNA sequencing.

While an amazing revolution, application of a technology such as DNA sequencing is limited by the size and cost of DNA sequencers, which in turn restricts accessibility. However, recent breakthroughs are allowing DNA sequencing to take place in jungles, the arctic, and even space—giving science the opportunity to reach further, faster than ever before. 

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra.

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra. Photo credit: Field Projects International.

The newfound accessibility of DNA sequencing means a marriage between fields of science that were previously largely unacquainted. The disciplines of genomics and wildlife biology/ecology have largely progressed independently. Wildlife biology is practiced in the field through observations and macro-level assessments, and genomics, largely, has developed in a lab setting. Leading the charge in the convergence of wildlife biology and genomics is Field Projects International. 

Continue reading “Deep in the Jungle Something Is Happening: DNA Sequencing”

Understanding Mechanisms of Pesticide Resistance to Thiamethoxam in the Cotton Aphid

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A. gossypii on cotton leaf. Image credit: Clemson University - USDA Cooperative Extension Slide Series, , United States [CC BY 3.0 (https://creativecommons.org/licenses/by/3.0)], via Wikimedia Commons
A. gossypii on cotton leaf. Image credit: Clemson University – USDA Cooperative Extension Slide Series, , United States [CC BY 3.0

The extensive and repetitive use of neonicotinoids has led to the development of resistance in several insect species including, the cotton aphid, A. gossypii. A. gossypii is a widely distributed pest that affects watermelons, cucumbers, pumpkin, cotton, and citrus crops, among others, making it one of the most economically important agricultural pests known. Thiamethoxam is a neonicotinoid insecticide that irreversibly binds to the nicotinic acetylcholine receptors (nAChRs) of cells in the nervous system and interferes with the transmission of nerve impulses in insects (1).

To further understand the mechanisms of resistance to thiamethoxam and other neonicotinoids, Wu et al. recently investigated (2) expression changes in the transcripts of P450 in thiamethoxam-susceptible and thiamethoxam-resistant cotton aphid strains. Nine P450 genes were significantly overexpressed in the resistant strain (especially CYP6CY14). The involvement of overexpressed P450s was examined through RNA interference (RNAi) introduced via artificial diet and dsRNA feeding.

Continue reading “Understanding Mechanisms of Pesticide Resistance to Thiamethoxam in the Cotton Aphid”

Cell Free Application: Characterization of Long Non-coding RNA Inhibition of Transcription

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Long noncoding RNAs have been shown to regulate chromatin states, transcriptional activity and post transcriptional activity (1). Only a few studies have observed long non-coding RNAs modulating the translational process (2). The noncoding RNA BC200 has been shown to inhibit translation by interacting with the translation initiation factors, eIF4A and eIF4B.

To characterize how BC200 translational inhibition could be controlled,  a variety of RNAs were transcribed/translated in vitro using the TNT system (Cat. #L4610) from Promega. To each transcription/translation reaction, BC900 RNA, hnRNPE1 and hnRNE2 proteins were added. Inhibition of BC200 activity was noted when proteins were successful expressed (3).

Literature Cited

  1.  Sosinska, P et.al. (2015) Intraperitoneal invasiveness of ovarian cancer from the cellular and molecular perspective. Ginekol. Pol. 86, 782–86.
  2. Geisler, S. and Coller, J. (2013) RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat.Rev. Mol. Cell. Bio. 14,699–12.
  3. Jang, S. et. al. (2017) Regulation of BC200 RNA-mediated translation inhibition by hnRNP E1 and E2. FEBS Letters. 591, 393–5.