GFP - Imaging Fluorescent Proteins

Techniques and Protocols

Molecular Biology

Clone your gene of interest into a (commercially available) XFP-containing vector (e.g. from Clontech). By doing so, generate either a vector for cotransfection of your gene of interest and GFP or a GFP-fusion protein or a fusion proteins. In case of a fusion protein the XFP-part can be linked N-terminally, C-terminally or be contained centrally in the protein sequence. Where you put it depends on the functional sides or your protein. Inserting the XFP at the wrong site may lead to a non-functional fusion protein. Check cloning result by sequencing.

Labelling of subcellular structures / organelles: pick a vector with a respective translocation sequence.

Promotor structure: do you want to have it inducible?

Vector: for transient or stable transfection? Selection marker for transfection?

Transfection

CaPhosphate, Electroporation, Lipofectamin a.o.
stable or transient? transgenic organisms?

Imaging

Seed cells on coverslips (in tissue plates with coverslip bottom), coat coverslips with Gelatine, Poly-L-Lysin, Fibronectin if necessary.

Cell fixation: 2-3% Formaldehyde, Triton-X100, Methanol
Store fixed cells with an antibleaching agent!

It is important to optimize the fluorescent filters of your microscope according to the XFP you are using. Only this guarantees optimal background-to-noise signal. When you are working with multiple labels in one cell, you can use double/trippel-filter sets. In combination with a monochromator as a light source you can very quickly image the different dyes without having to change the filter set.