Ultimately, and the breast cancer cells that went untreated

Ultimately, both green florescent proteins tagging and
polyhistidine tagging are both useful to monitor and track specific gene
activity. Both have their pros and cons as methods of immobilized
metal-affinity chromatography, but with further research there can be improvements
in both techniques to improve the tracking of genes and protein-protein
interactions over time.

In research done by Coumans et al, human breast cancer cells were
analyzed using green fluorescent proteins to track proteome changes. Breast
cancer cells were cultured and treated with antibiotics under restricted
conditions and were then tagged with GFP (Coumans et al, 2014). The proteins were extracted after grown to a set size
and analyzed using two-dimensional electrophoresis and isobaric tags for
relative and absolute quantitation (iTRAQ) (Coumans et al, 2014).  Comparisons
were made on cell behaviour and protein-protein interactions between the cells
that were treated with antibiotics and tagged with GFP and the breast cancer
cells that went untreated (Coumans et al,
2014). It was found that the antibiotic improved the outcome of the cells,
but many results came up inconclusive (Coumans et al, 2014). There was proof of analytical variability and
evidence of molecular changes caused by the GFP expression within the cell (Coumans
et al, 2014).

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Within a protein, GFP can be fused to either the N or
C terminus or between two proteins to study the protein-protein interactions but
the insertion must be done strategically so there is a lack of disruption to
the natural reading frame (Catanzariti et
al, 2004). Due to its fluorescence,
scientists can isolate where the gene is expressed and when GFP is triggered by
blue ultraviolet light and produces a green fluorescent light. When a light
with a wavelength of 400nm strikes the chromophore, the protein goes into an
excited state (Heim et al, 1994). When
the chromophore enters ground state, the light emitted will be at the
wavelength 505nm (Heim et al, 1994).
This light appears on the visible light spectrum as a green colour (Heim et al, 1994).

The green fluorescent protein (GFP) is a bioluminescent
protein that was first discovered within Aequorea
victoria, which is a species of jellyfish (Prasher et al, 1992). The green fluorescent protein works be producing a
fluorescent glow in vivo allowing scientists to study the dynamics of the
protein under a microscopy without altering or killing the cell (Tsien, 1998). Within
a plasmid, GFP can be placed subsequent to the promoter region and beside the
gene of interest. The restriction enzymes can cut the section of interest out
and then add the enzyme ligase and then the plasmid can be added to a protein (Tsien,
1998).  

Histidine tagging is a method of IMAC that is very
simple to use and can achieve a significant increase of purification in a
single step (Kaake et al
2010). The polyhistidine residue must
first be fused to the gene of interest within a plasmid (Singh and Jain, 2013).
The plasmid must be capable of replication, contain a gene available for the
allowance that only select cells can accept the plasmid, and have a multiple
cloning site (MCS) so that is can be recognized by restriction enzymes (Singh and Jain, 2013). The lac operon
creates sites that manage when the histidine-tagged protein is expressed as
well as gives a site for repressor proteins to bind to (Singh and Jain, 2013). The
ribosome then begins translation which covers the histidine tagged location
from the start codon until the stop codon (Singh and Jain 2013). Once
translated, the protein must be purified (Porath, 1992).
The protein must go through resin-bound matrix that contains metal ions so that
the histidine residues can transfer electrons to form an immobilized chelate
complex (Porath, 1992). Finally, the
protein must be eluted (Bornhorst and Falke, 2000). There are two methods to
elute the polyhistidine-tagged protein (Bornhorst and Falke, 2000). One way is to increase the pH so that the imidazole
ring of the histidine side will become protonated and the bonds that connect
the immobilized metal ion and the histidine side chain will break (Bornhorst
and Falke, 2000). This will lead to the
protein eluting from the matrix (Bornhorst and Falke, 2000). The second way is to use free imidazole to elute the
protein (Bornhorst and Falke, 2000).
The imidazole will help the histidine bind to the metal (Bornhorst
and Falke, 2000).. Histidine tagging
is beneficial because it identifies protein-protein interactions, but does not
affect the function of the protein that it is involved with (Kimple et al, 2013). His-tagged proteins are easily detected and the ions
that the residues can bind to are stable, specifically in acidic conditions (Kimple et al, 2013). The polyhistidine tag can have a range of two to ten
histidine residues, but the most commonly seen tag consists of six residues (Kimple et al, 2013). These residues are typically found on either the N
or the C terminus (Porath 1992).

Immobilized metal-affinity chromatography (IMAC) is a
technique used to purify a protein and isolate various properties of that
protein (Kaake et al
2010). Using specific tags to track
certain characteristics of a protein is a straightforward way to analyze
certain genes and protein-protein interactions. Histidine tagging and Green Fluorescent
Protein (GFP) are two distinct types of peptide sequences that act as a protein
tag. The purpose of these protein tags is to enable the detection and
purification of the expressed proteins and the inherent binding in which there
could be a protein-protein interaction.