Fluorescent proteins & dyes
A green or red fluorescent protein fused in frame to the protein of interest and expressed in the cells, so its location and movement can be followed live. The choices are which protein, which end, which promoter, and how to keep two colours apart.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
GFP / mCherry fusions
A green or red fluorescent protein fused in frame to the protein of interest and expressed in the cells, so its location and movement can be followed live. The choices are which protein, which end, which promoter, and how to keep two colours apart.
How it differs from the standard method
- Choose a bright, monomeric, fast-maturing protein (mNeonGreen or mEGFP; mCherry or mScarlet) over older ones (ECFP, DsRed).
- Tag the end that does not carry a targeting signal; add a short flexible linker.
- Express at near-endogenous levels and image sequentially when using two colours.
1Two ways to make something glow
A fluorescent label is either genetically encoded or chemical. A fluorescent protein (GFP and its relatives) is fused to the protein you want to follow and made by the cell itself, so it reports location and dynamics in living cells without any staining step.
EC × QY
How many photons a label gives is its extinction coefficient times its quantum yield, then times how well your laser line and filters match its spectra. mNeonGreen is about three times as bright as EGFP; mCherry about half as bright.
Monomers only
Early red proteins such as DsRed are obligate tetramers and EGFP dimerises weakly at high local concentration. Fused to a protein, an oligomeric tag cross-links its partners into clumps.
Time to glow
A fluorescent protein must fold and form its chromophore before it lights up: minutes for mNeonGreen or mCherry, many hours for DsRed, which passes through a green intermediate on the way.
Bleed-through
Emission spectra have long red tails. A green protein excited at 488 nm sends a few percent of its light into a red channel, which matters if both channels are captured at once.
2Where the tag goes
A fluorescent protein is about 27 kDa, bigger than many of the proteins it labels. Put it on the end that matters least for function and targeting: proteins anchored by a C-terminal lipid (Rab and Ras GTPases, CAAX-box proteins) must be tagged at the N-terminus, and signal peptides at the N-terminus force a C-terminal or internal tag.
3DNA and organelle dyes
Hoechst 33342 is membrane-permeant and stains live nuclei within minutes; DAPI crosses intact membranes poorly, so it is the dye for fixed cells and, in live cultures, a marker of dead cells. Both are excited in the UV, and UV light on DNA loaded with a dye damages it, so live imaging uses the lowest concentration and dose that still segment the nuclei.
Key terms
Practise these as flashcards →Extinction coefficient × quantum yield; the photons a fluorophore gives per unit of excitation.
A fluorescent protein engineered not to dimerise (e.g. mEGFP with A206K, mCherry), so it does not cross-link the protein it tags.
Folding and chromophore formation after translation; until it is complete the protein is not fluorescent.
Signal from one fluorophore detected in another's channel, usually from the red tail of the emission spectrum.
Irreversible destruction of the fluorophore by light; each protein and dye has a characteristic photostability.
A lipophilic cation, such as MitoTracker or TMRM, that concentrates in mitochondria in proportion to their potential.
A general stain, typically of nuclei, that shows where every cell is so specific labels can be counted per cell.
When to use it
- Following a protein's location and dynamics in living cells over minutes to days: a fusion to a monomeric fluorescent protein.
- Marking every nucleus for counting, tracking or segmentation: Hoechst 33342 in live cells, DAPI in fixed ones.
- Showing the mitochondrial network, or comparing membrane potential between conditions: MitoTracker loaded live, with an uncoupler control.
Compared with alternatives
Labels endogenous proteins in fixed cells with antibodies; use it to check a fusion localises like the native protein.
The microscope most labels are first imaged on; its filters decide which labels can be combined.
Adds environmental control and dose planning when the labelled cells are followed for hours.
Measures label brightness in thousands of cells without images; good for sorting cells to a low, uniform expression level.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A green or red fluorescent protein fused in frame to the protein of interest and expressed in the cells, so its location and movement can be followed live. The choices are which protein, which end, which promoter, and how to keep two colours apart.
- Choose a bright, monomeric, fast-maturing protein (mNeonGreen or mEGFP; mCherry or mScarlet) over older ones (ECFP, DsRed).
- Tag the end that does not carry a targeting signal; add a short flexible linker.
- Express at near-endogenous levels and image sequentially when using two colours.