Madison, WI is home for Promega, and while it is not a huge city, Madison is home to many biotech companies, fed mostly by the local, world-class University of Wisconsin-Madison. Many scientists and scientist families work, live and play near one another here. It is not uncommon for two scientists from different companies to talk to one another and discover that their respective companies have products or processes that could benefit both companies.
Case in point: Scientists at Promega have a good working relationship with Cellular Dynamics International (CDI), a biotech firm that specializes in differentiated iPSC-derived cells. We want to demonstrate that our assays work in iPSC cells and CDI wants to demonstrate the range of assays that can be performed with their iPSC-derived cells.
Most, if not all, processes within a cell involve protein-protein interactions, and researchers are always looking for better tools to investigate and monitor these interactions. One such tool is the protein complementation assay (PCA). PCAs use a reporter, like a luciferase or fluorescent protein, separated into two parts (A and B) that form an active reporter (AB) when brought together. Each part of the split reporter is attached to one of a pair of proteins (X and Y) forming X-A and Y-B. If X and Y interact, A and B are brought together to form the active enzyme (AB), creating a luminescent or fluorescent signal that can be measured. The readout from the PCA assay can help identify conditions or factors that drive the interaction together or apart.
A key consideration when splitting a reporter is to find a site that will allow the two parts to reform into an active enzyme, but not be so strongly attracted to each other that they self-associate and cause a signal, even in the absence of interaction between the primary proteins X and Y. This blog will briefly describe how NanoLuc® Luciferase was separated into large and small fragments (LgBiT and SmBiT) that were individually optimized to create the NanoBiT® Assay and show how the design assists in monitoring protein-protein interactions.
One piece of advice you will get from our Technical Services and R&D Scientists with regard to cell-based assays is to pay attention to what you are doing. Sounds obvious, but sloppiness can easily enter into the equation. Do you always count your viable cells with a hemocytometer and trypan blue exclusion before you split a culture? Do you always make sure that each well of your plate or plates contain the same number of cells? Two of our scientists, Terry Riss and Rich Moravec, published a paper demonstrating how decisions you make in experimental setup can ultimately affect the results you obtain. A natural consequence of this is difficulty replicating experiments if you didn’t pay attention to the details during the initial experimental setup.
Cell Density Per Well Affects Response to Treatment
To demonstrate how cell density can affect your data, Riss and Moravec set up parallel plates with three different cell densities of HepG2 cells and measured the response to tamoxifen. The lower the cell density per well, the more pronounced the effect of the tamoxifin on the cells. Higher density cells were more resistant to tamoxifen. Continue reading “Take Notes and Graduate Faster!”
Transient transfection is often used to perform reporter assays. We have advocated using a dual-reporter system for decades to normalize the data obtained and gain a clearer understanding of your results. The experimental reporter should vary with treatment and the control reporter should vary little with treatment. The control reporter thus serves as a marker to help you understand the relative activity of your experimental reporter. The bioluminescent Dual-Luciferase® method allows for sequential detection of the second reporter in a single sample providing a simple two-step normalization method. Here are seven ways in which dual-reporter assays help you avoid misinterpreting results.
Simply comparing the ratio of the experimental to the control reporter can resolve differences in:
Number of Cells/Well: When manually pipeting cells into a 96-well plate, there is always a chance of having variable numbers of cells in each well. This variation is cell number will affect the experimental and control reporters equally, so the ratio of experimental:control reporter activity will eliminate false interpretation of the experimental data–whether it affects an entire row or column on the plate or individual wells.
Transfection Efficiency: The variations in transfection efficiency will equally affect both the experimental and control reporters so the ratio of activity in dual-reporter assays will normalize the data.
Cell Viability: Often, reporter assays look at the dose response curve of a particular compound with regard to gene expression. Ideally, if a compound causes a change in the experimental reporter the control reporter will demonstrate little effect. However, if the compound is toxic, both the experimental and control will be altered and the ratio will tell you whether the compound truly affects reporter activity or just kills the cells.
Lysis Efficiency: When lysing a plate of cells, you could encounter situations where rows or columns lyse differently, especially if you are using manual disruption or get interrupted mid-plate. The difference is lysis will affect the experimental and control equally so the ratio will remove the variation.
Temperature: Ideally, a plate should be equilibrated to ambient room temperature before proceeding to the reporter assay. Plates can cool at different rates or researchers anxious to record data may read the data early. Temperature variations will affect both reporters so the ratio will limit the affect on the data.
Measurement Time: Repetition of data is a hallmark of good science. You are often called upon to repeat experiments sometimes days or weeks apart. Let’s say you repeat your experiment one week after the initial experiment. The first time you measured the response, you waited 10 minutes after reagent addition to read, this week you waited 30 minutes. This will affect both reporters equally and therefore the ratio will allow you to more easily compare the data from this week and last week.
Bonus Benefit from Dual-Luciferase®, Dual-Glo® and the NanoGlo® Dual Luciferase Reporter Systems: NoLysate Splitting: Promega dual-reporter assays are designed for same-well multiplexing so there is no chance of variations creeping into your data due to unequal splitting of the cellular lysate to measure two separate reporter activities.
Since the introduction of the first bioluminescent dual-luciferase assay in 1995, this approach has been used in countless studies to advance our scientific understanding of cellular gene regulation. To learn more about the last 30 years of bioluminescent innovations and research discoveries please visit our 30th anniversary web page.
By clicking “Accept All”, you consent to the use of ALL the cookies. However you may visit Cookie Settings to provide a controlled consent.
If you are located in the EEA, the United Kingdom, or Switzerland, you can change your settings at any time by clicking Manage Cookie Consent in the footer of our website.
Necessary cookies are absolutely essential for the website to function properly. These cookies ensure basic functionalities and security features of the website, anonymously.
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics".
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other.
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertisement".
This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance".
6 months 2 days
This cookie is set by the provider Media.net. This cookie is used to check the status whether the user has accepted the cookie consent box. It also helps in not showing the cookie consent box upon re-entry to the website.
This cookie is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
Analytical cookies are used to understand how visitors interact with the website. These cookies help provide information on metrics the number of visitors, bounce rate, traffic source, etc.
This cookie is associated with Sitecore content and personalization. This cookie is used to identify the repeat visit from a single user. Sitecore will send a persistent session cookie to the web client.
This domain of this cookie is owned by Vimeo. This cookie is used by vimeo to collect tracking information. It sets a unique ID to embed videos to the website.
1 month 18 hours 24 minutes
This cookie is used to calculate unique devices accessing the website.
This cookie is installed by Google Analytics. The cookie is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. The cookies store information anonymously and assign a randomly generated number to identify unique visitors.
This cookie is installed by Google Analytics. The cookie is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visted in an anonymous form.
Advertisement cookies are used to provide visitors with relevant ads and marketing campaigns. These cookies track visitors across websites and collect information to provide customized ads.
1 year 24 days
Used by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
This cookie is set by doubleclick.net. The purpose of the cookie is to determine if the user's browser supports cookies.
5 months 27 days
This cookie is set by Youtube. Used to track the information of the embedded YouTube videos on a website.
Performance cookies are used to understand and analyze the key performance indexes of the website which helps in delivering a better user experience for the visitors.
This cookies is set by Youtube and is used to track the views of embedded videos.
This is a pattern type cookie set by Google Analytics, where the pattern element on the name contains the unique identity number of the account or website it relates to. It appears to be a variation of the _gat cookie which is used to limit the amount of data recorded by Google on high traffic volume websites.