For Protein Complementation Assays, Design is Everything

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.

Continue reading “For Protein Complementation Assays, Design is Everything”

Take Notes and Graduate Faster!

Cell density illustrationOne 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!”

2 Ways to Save Your Single Reporter Data

Reporter assays using a single reporter, be it from a stable cell line or transient transfection, can benefit from normalization. Obviously, we are not talking about adding a second control reporter but normalizing to the number of live or dead cells in the well.  Two cell health assays, CellTiter-Fluor™ Cell Viability Assay and CellTox™ Green Cytotoxicity Assay, are ideally suited for multiplexing with reporter assays.  Continue reading “2 Ways to Save Your Single Reporter Data”

6 + 1 Ways Dual-Reporter Assays Can Save Your Data

Dual-Reporter-Assay

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. 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:

  1. 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.
  2. Transfection Efficiency: The variations in transfection efficiency will equally affect both the experimental and control reporters so the ratio will normalize the data.
  3. 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.
  4. 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.
  5. Temperature: Ideally, a plate should be equillibrated 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.
  6. 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: Lysate 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.

Further Reading:
Normalizing Genetic Reporter Assays Approaches and Considerations for Increasing Consistency and Statistical Significance