Why You Don’t Need to Select a Wavelength for a Luciferase Assay

It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.

Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest. Continue reading “Why You Don’t Need to Select a Wavelength for a Luciferase Assay”

What Makes a “Good” Buffer?

Buffers are often overlooked and taken for granted by laboratory scientists, until the day comes when a bizarre artifact is observed and its origin is traced to a bad buffer.

The simplest definition of a buffer is a solution that resists changes in hydrogen ion concentration as a result of internal and environmental factors. Buffers essentially maintain pH for a system. The effective buffering range of a buffer is a factor of its pKa, the dissociation constant of the weak acid in the buffering system. Many things, such as changes in temperature or concentration, can affect the pKa of a buffer.

In 1966, Norman Good and colleagues set out to define the best buffers for biochemical systems (1). By 1980, Good and his colleagues identified twenty buffers that set the standard for biological and biochemical research use (2,3).  Good set forth several criteria for the selection of these buffers: Continue reading “What Makes a “Good” Buffer?”

Meet the Mighty Masked Masters of Measurement: #WorldMetrologyDay

Scientific investigation is an iterative process, for which reproducibility is key. Reproducibility, in turn, requires accuracy and precision—particularly in measurement. The unsung superheroes of accuracy and precision in the research lab are the members of your local Metrology Department. According to Promega Senior Metrologist, Keela Sniadach, it’s good when the metrology department remains unsung and behind the scenes because that means everything is working properly.

Holy Pipettes, Scientists! We have a metrology department?! Wait…what’s metrology again?

Callibration technician checks out a multipipettorMetrology (the scientific study of measurement) got its start in France, when it was proposed that an international length standard be based on a natural source. It was from this start that the International System of Units (SI), the modern metric system of measurement, was born.

Metrology even has its own day: May 20, which is the anniversary of day the International Bureau of Weights and Measures (BIPM) was created by the Meter Convention in Paris in 1875. The job of BIPM is to ensure worldwide standards of measurement.

For life scientists, metrology centers around making sure the equipment used everyday—from pipettes to heating blocks to centrifuges—is calibrated and measuring correctly. Continue reading “Meet the Mighty Masked Masters of Measurement: #WorldMetrologyDay”

Eight Considerations for Getting the Best Data from Your Luminescent Assays

The stage is set. You’ve spent days setting up this experiment. Your bench is spotless. All the materials you need to finally collect data are laid neatly before you. You fetch your cells from the incubator, add your detection reagents, and carefully slide the assay plate into the luminometer. It whirs and buzzes, and data begin to appear on the computer screen. But wait!

Bad data
These data are garbage!

Don’t let this dramatic person be you. Here are 8 tips from us on things to watch out for before you start your next luminescent assay. Make sure you’ll be getting good data before wasting precious sample!

Continue reading “Eight Considerations for Getting the Best Data from Your Luminescent Assays”

Converting RPM to g Force (RCF) and Vice Versa

Radius

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”

Lab Sustainability Doesn’t Have To Be Painful

Ian Nicastro says he didn’t set out to start a green revolution.

“I’m not hardcore ‘Save the trees,’” Ian says. “I’m probably a little different from the people you traditionally see as promoting the sustainability thing. Obviously, I do want to help the environment, but for me it was like, ‘this is logical, and we should be doing this.’”

Ian is the lab manager of the Pasquinelli Lab, a C. elegans lab at the University of California–San Diego that studies miRNA and its role in processes like aging. He’s been in the lab for about six and a half years, splitting his time between research and lab management duties. According to Allison Paradise, the CEO of My Green Lab, Ian has put out some “outstanding” efforts to implement sustainable practices in the lab. Continue reading “Lab Sustainability Doesn’t Have To Be Painful”

Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?

Confused womanI’ve got a set of experiments planned that, if all goes well, will provide me with the answer I have been seeking for months. Plus, my supervisor is eagerly awaiting the results because she needs the data for a grant application, so I don’t want to mess it up. However, I am faced with a choice for my firefly and Renilla luciferase reporter assays: Do I use the Dual-Luciferase® Reporter Assay System or Dual-Glo® Luciferase Assay System? What’s the difference? How do I decide which to use? I’m so confused! Help!

Sound familiar? Not to worry! The choice is not difficult once you know how these assays work and how they differ.
Continue reading “Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?”

A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs. Continue reading “A BiT or BRET, Which is Better?”

It’s Almost iGEM Season—Help Is On The Way!

The 2019 iGEM Competition is on the horizon and team registration opens this month. We’re excited to partner with the iGEM Foundation again this year and offer our support to the young scientists who participate. If you’re starting an iGEM project, there are going to be things you need along the way. We are pleased to share a number of different ways we can help your iGEM team from now until the Giant Jamboree.

Grant Sponsorship

Tell us about your iGEM project and your team could win a 2019 Promega iGEM Grant Sponsorship. Ten winning teams will each receive $2000 in free Promega products to use for their iGEM projects. Tell us about your project—What problem are you addressing? What is your proposed solution? What challenges does your team face? Last year’s winning teams selected from a wide range of reagents and supplies, including master mix, restriction enzymes, ligase, DNA purification kits, expression systems, DNA ladders and markers, buffers and agarose. Click here to apply! Continue reading “It’s Almost iGEM Season—Help Is On The Way!”

Executing a NanoBRET™ Experiment: From Start to Data

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”