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Fluorescent Protein Tagging Techniques

Fluorescent proteins like GFP can be used as tags to monitor gene expression and protein interactions in cells. Specific applications include fluorescence resonance energy transfer (FRET) to observe if two labeled proteins interact, photoactivation to track protein movement, and fluorescence recovery after photobleaching (FRAP) to study protein diffusion and transport rates. Affinity chromatography also uses tags, like histidine or fusion proteins, to purify specific proteins by binding them to affinity matrices then eluting them with salt or pH changes. Radioisotope labeling incorporates radioactive markers to trace small molecules and distinguish between chemically identical molecules.

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Ronnie Kot
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13 views3 pages

Fluorescent Protein Tagging Techniques

Fluorescent proteins like GFP can be used as tags to monitor gene expression and protein interactions in cells. Specific applications include fluorescence resonance energy transfer (FRET) to observe if two labeled proteins interact, photoactivation to track protein movement, and fluorescence recovery after photobleaching (FRAP) to study protein diffusion and transport rates. Affinity chromatography also uses tags, like histidine or fusion proteins, to purify specific proteins by binding them to affinity matrices then eluting them with salt or pH changes. Radioisotope labeling incorporates radioactive markers to trace small molecules and distinguish between chemically identical molecules.

Uploaded by

Ronnie Kot
Copyright
© All Rights Reserved
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Tag - Protein, Youre It!

1.

Colored Tags

use fluorescent proteins (ex: Green Fluorescent Protein, GFP, which originates in jellyfish)

GFP can be used as a reporter molecule


monitor gene expression

ex. Cat treated with the GFP gene

Applications for colored tags:


1. Fluorescence Resonance Energy Transfer
(FRET)
2 molecules of interest are labeled with different
fluorochromes that one fluorochrome spectrum
overlaps with the absorption spectrum of the other
if the two proteins bind together and fluorochromes
are close together, one fluorochrome transfers
energy of absorbed light to the other
Why is this important?- able to see if certain
proteins bond with each other
2.
Photoactivation
synthesizing an inactive fluorescence molecule of
interest, introducing it to a cell, and then activating
it suddenly at a chosen site by focusing light on
that area
Why is this important?- able to observe how a
protein moves and is organized in a cell
ex: tubulin labeled with caged fluorescein- can be
incorporated into microtubules of mitotic spindles, and when the tubulin becomes fluorescent,
scientists are able to track its movement along microtubule
3. Fluorescence Recovery After Photobleaching (FRAP)

2.

4.

blasting of strong, focused beam of light from a laser to extinguish GFP protein fluorescence in
specific region of a cell
by analyzing the way the rest of the fluorescent molecules move into bleached area, you can
obtain information about kinetic parameters
What can this be used for?- can give data on diffusion coefficients, active transport rates, or
binding and dissociation rates from other proteins
Affinity Chromatography
Method used to separate specific proteins and their recognition tags
Use of an insoluble matrix that is covalently bonded to a specific ligand that will bind to a
specific protein to then elude that protein by dissociating the complex with concentrated salt
solutions of varying pHs
Epitope - antigenic determinant recognition tag that is recognized by its specific molecule (i.e.
antibody)
Examples of affinity chromatography include:
metal affinity: the amino acid histidine binds to certain metal ions (Ni and Cu ions) and the
modified protein can be retained on the column of this technique which then contains the
immobilized nickel or copper ions
fusion protein affinity: entire protein acts as a recognition tag where cells are made to
synthesize an enzyme and a fusion protein is then formed and purified separately from the rest
of the molecules, but still remains binded strongly to the enzyme which occurs normally in
nature
this is then followed by a cleaving the protein of choice and the recognition tag by an
engineered amino acid sequence without destroying the purified protein
this cleavage is known as tandem affinity purification tagging or tap-tagging
Other matrices used for chromatography are ion-exchange and gel-filtration, although affinity is
discussed solely for its efficiency in procedure
Radioisotope - by using chemical markers to incorporate into the synthesis of a small molecule,
the fate of that molecule and the specific atoms that make it can be traced during any biological
reaction.
This technique can also distinguish chemically identical molecules with different histories
(e.g. those that differ in time of synthesis).
It can also be combined with gel electrophoresis to determine protein sizes.

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