How do you visualize gfp

The original expression plasmid for this jellyfish protein, green fluorescent protein GFP yielded a low level of fluorescence. However, through mutagenesis, enhanced GFPs have been produced that are about 35 times brighter than the wildtype GFPs 3 , 4. There are now blue and yellow fluorescent proteins and other colors may soon be available.

The size of green fluorescent protein, about 28 kD 5 , 6 , creates a large fluorescent tag in comparison to the Dalton fluorescent dyes that have been coupled to purified proteins; nevertheless, GFP does not seem to interfere with most interactions of its linked protein. There are cases, however, where the placement of the large GFP probe has interfered with the nearby domains of proteins. For example, coupling GFP to the N-terminal region of alpha-actinin, near the actin-binding domain resulted in a GFP probe that could not bind actin, whereas GFP linked to the C-terminus of alpha-actinin resulted in a probe that readily bound actin filaments 7.

An alternate approach to eliminating interference by GFP is to introduce a linker of several amino acids between the GFP probe and the protein 8. The expression of GFP-linked proteins has been particularly advantageous for following proteins whose low abundance or solubility properties make them unsuitable for microinjection into living cells.

The cDNAs for a number of abundant cytoskeletal proteins have also been. Springer Nature is developing a new tool to find and evaluate Protocols. Learn more. Skip to main content. This service is more advanced with JavaScript available. Advertisement Hide. Authors Authors and affiliations Joseph C. Ayoob Jean M. Sanger Joseph W. Terms of Service. Previous Section Next Section. Equipment Microscope either dissecting or inverted compound with fluorescent visualization capabilities Tissue culture dishes or glass-bottomed tissue culture dishes MatTek Corporation or multichamber culture plates Nalgene Lab-Tek 2 Chambers.

Previous Section. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci 97 : — Gene : 33 — CrossRef Medline Google Scholar.

A guide to choosing fluorescent proteins. Nat Methods 2 : — Tsien RY. The green fluorescent protein. Annu Rev Biochem 67 : — Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J Biol Chem : — Plant Physiology, 1 , — Real-time dynamics of peptide ligand-dependent receptor complex formation in planta.

Science Signaling, 8 , ra This website uses cookies. Those have two functions: On the one hand they are providing basic functionality for this website. On the other hand they allow us to improve our content for you by saving and analyzing anonymized user data. You can redraw your consent to using these cookies at any time. Find more information regarding cookies on our Data Protection Declaration and regarding us on the Imprint.