Getting the most from fluorescent proteins, Part 2

On Feb 3^rd in our previous blog entry on fluorescent proteins, we discussed some basic tips on setting yourself up for success with fluorescent protein based experiments. Here are some more ideas to help boost your imaging success:

1.    Know your background.

All cells contain endogenous fluorescent materials which can confound image interpretation, especially when your fluorescent protein signal is weak.  Make sure you’re familiar with the autofluorescence of your cell type before starting your FP experiments: take some images of non- expressing control cells using the same filters and excitation wavelength as you plan to use for your FP imaging.

Keep in mind that for mammalian cells, autofluorescence is confined mainly to the blue and green regions of the visual spectrum, while in other organisms (e.g. plants, yeast, and bacteria), some cell types may contain fluorescent compounds in other regions of the spectrum. For any given species and cell type, there is likely to be a wavelength “window” with the least autofluorescence; try to choose a fluorescent protein in this wavelength range for maximum signal above background.

2.    Sometimes two (or more) FPs are better than one.

If you are having trouble obtaining sufficient fluorescent signal from a fluorescent protein fusion construct, consider adding an extra copy of the fluorescent protein to boost your brightness.  While this is not recommended unless all else has failed, for low-abundance proteins it can substantially increase the likelihood of detection.  It is possible to create a functional fusion of two or more copies of fluorescent protein in many cases, although the larger size of such a tag increases the chances of mislocalization, so proper controls and validation are essential if you use this technique.  Also, remember that it is generally difficult to use PCR to amplify tandem copies of any gene, including FPs, so restriction-based subcloning is the most reliable way to create multi-copy FP tags.

3.    The best fluorescent proteins don’t stick together!

Truly monomeric fluorescent proteins make the best fusion tags, since they don’t produce localization artifacts due to multimerization. Even weak dimers, such as EGFP and its derivatives, can cause trouble if your fusion protein is at high concentration or in a confined space like a membrane or vesicle.

Are you still using your old EGFP fusion constructs?  If so, make sure to validate your localization results by other methods, or switch to a truly monomeric FP such as mTFP1 or mWasabi.  If you prefer to keep your original constructs, note that any Aequorea GFP-derived FP can be made completely monomeric by adding the A206K mutation.

One final warning — many commercially available FPs that were initially advertised as being monomeric later turned out to be dimers!  With any new FP you try, validate your results before making your conclusions.

Tags: cell marker, cellular, cellular localization, fluorescence, fluorescent protein, FRET, GFP, imaging, mTFP1, mWasabi

Getting the most from fluorescent proteins

Fluorescent proteins (FPs) are an indispensable component of the biology toolbox, providing a robust and straightforward method to optically label nearly any protein of interest.

While most FPs can be used for a wide variety of experimental setups and conditions, getting the best quality data from your hard efforts requires some forethought. Here are a few tips to get the most out of FP imaging:

1.Reduce pre-measurement photobleaching.

All FPs photobleach upon exposure to excitation light. Some, like commonly used YFPs, bleach rather quickly, while others, such as Allele’s mTFP1, are substantially more photostable. However, even the most photostable FPs can be susceptible to excessive pre-measurement bleaching if precautions are not taken.

While searching for your favorite cell on the microscope, try to use the lowest possible excitation light intensity. Close the shutter when you’re setting up software or other experimental apparatus, and use short exposure times whenever possible during focusing.

2.Consider pH and other variables.

Most FPs are somewhat sensitive to acidic pH. Some, such as mTFP1 and many of the red FPs, are reasonably resistant to pH changes, while others, such as EYFP, are highly sensitive. If you’re imaging acidic compartments such as lysosomes or plant vacuoles, you’re unlikely to see any fluorescent signal if you’re using EYFP or any other pH-sensitive FP, so choose wisely!

Information on the fluorescence pKa (the pH at which 50% of the fluorescence emission is quenched) of new fluorescent proteins is generally easy to find, so do your homework!

3.Be careful with fusions and linkers.

One big advantage of using FPs is that they may be genetically fused to virtually any protein of interest. While FPs usually have a negligible effect on the properties of their fusion partners, it’s always a good idea to double-check and validate data on new proteins.

If you don’t know where your protein should localize, check both N- and C-terminal FP fusions to be sure they give the same results. If not, validate your localization by other methods, such as antibody staining. If you can devise a functional assay for your FP-labeled protein, this is also a good way to be sure the fusion isn’t causing trouble.

If you’re having problems with a particular FP fusion, try a few different linkers between the FP and the protein of interest. Floppy linkers, such as poly-(Gly-Gly-Ser-Gly-Gly-Thr) frequently work well, but occasionally rigid linkers (such as poly-proline) or other sequences will give better results. Unfortunately, the process of optimizing a fusion construct is largely empirical.If you can put in the effort early in your experiments to produce the best possible FP fusion, you’ll benefit greatly in later experiments!

Tags: cell imaging, cellular localization, cellular marker, fluorescence microscopy, fluorescent microscopy, Fluorescent proteins, FPs, FRET