Analysis of Chimeric Proteins by

Fluorescence Microscopy and

Western Blotting
By Connor Thornton
Keith Herrera, Kristina Wright, Dhruv Patel
Section 9

IntroductionThe cytoskeleton is a network of proteins only found in all eukaryotic cells

and in many bacterial cells and provides cell shape and stability as well as providing
pathways of transport and signaling within the cell. The cytoskeleton is composed of
three parts, an actin based network, a microtubule network comprised of tubulin
and a network of various intermediate filament type proteins (Vats et al, 2009).
Microtubules begin near the center of the cell in a complex called the microtubuleorganizing center and extend outwards (Pollenz et al, 2008).
Chimeric proteins are spliced genes caused by the mismatching of coding genes in
a DNA or RNA sequence during replication. This causes the mismatched genes to be
coded together creating a new gene. This method can be manipulated using
plasmid DNA so that the incorporated gene would be produced along with the host
gene (Herrera-Estrella, 1983). In this case a gene coding for the production of a
tagging molecule is attached to several genes in order identify their presence with
the addition of the nocodazole enzyme which prevents the polymerization of
microtubules.
GFP was found by Shimomura O., Johnson FH. and Saiga Y. from the jellyfish
Aequorea victori. GFP is a chemiluminescent protein that alone can act to produce
the posttranslational synthesis of the chromophore allowing it to illuminate in nonjellyfish organisms (Tsien, 1998). The GFP protein has 238 amino acids and weighs
27 KDa and is one of the most common chromatic tags as it can be fused with host
proteins without inhibiting either of the proteins. This allows the tagging and
identification of selected structures such as actin, tubulin, golgi and mitochondrial
proteins.
By preforming a SDS-PAGE followed by a Western Blotting of four different
chimeric proteins of a combination of EYFP and either golgi, actin, tubulin, or
mitochondrial and treated with an EYFP antibody then an alkaline phosphatase 2nd
degree antibody will allow for the identification of the location of EYFP on each
protein.

MethodsSDS-PAGEA SDS-PAGE was run using an 18% gradient Bio-rad Criterion Precast 10%
tris-HOL gel. The gel was submerged in 1x running buffer of 10% TRIS and loaded
with 20 g of protein in each lane with a Biorad Precision Plus Kaleidoscope
molecular marker. The gel was run for 50 minutes at 230 V.
Western BlotOnce complete the gel was removed and placed in a western blot sandwich
which was composed of a Scotchbrite Pad and blotting paper on top and bottom
with the gel and a nitrocellulose membrane in the middle. The sandwich was

compressed and submerged in 8% TRIS transfer buffer. The blot sandwiches were
electrophoresed at 120 mA for 50 minutes then removed and disassembled.
Antibody StainingThe nitrocellulose membrane was then treated with blocking solution of TBST
and 5% dry milk and incubated for 25 minutes. The nitrocellulose was then removed
and placed in EYFP antibody, Living Colors Arth-EYFP 100 L/10mL milk-TBST, for 45
minutes. Next, the nitrocellulose was washed in TBST three times for eight minutes
each then placed in a secondary antibody of alkaline phosphatase, Jackson
Laboratories Anti-Rabbit IgG w/ Alk plus 10 L/mL Milk-TBST, which binds to the
EYFP antibody for 35 minutes. It was then washed three times in TBST for 8 minutes
each and washed in TBS. Afterwards it was placed in NBT/BCIP to develop, rinsed
and then placed on blotting paper.
Computer analysisThe nucleotide sequences for each of protein chimera as well as the GFP
gene were run through the MW/pl calculator on the web.expasy.org website to find
the molecular weight and then again through blast.ncbi.nlm.nih.gov to find the
identities and similarities of each in order to identify the deviations and to
determine the location of the EYFP gene in each chimera.

ResultsThe molecular weights of each of the protein sequences were found using the
formula y=1.0327e-.013x where y is the Rf and x is the molecular weight as shown in
table 2.
Figure 1: Western Blot showing the migration of sequences with EYFP attached.
The first lane is the mitochondrial proteins, the second is golgi proteins, the third is
actin proteins and the fourth is tubulin proteins.

Table 1: Table of the molecular weight and its Rf that is graphed in figure 2 and is
used to calculate the molecular weight of the protein sequences.
M
W

Distance
(cm)

Rf
0.7
0
0.5
5
0.2
4
0.1
7

37 11.85/16
50 9.4/16
10
0 4.0/16
15
0 2.9/16

Figure 2: Graphic representation of table 1. The formula of the exponential trend

line is used to calculate the molecular weight of the protein sequences in figure 1.

Molecular Weight
1.00
20

40

60

80

100

120

140

160

f(x) = 1.03 exp( -0.01 x )

R = 0.96
Rf

0.10
Molecular Weight (g)

Table 2: Shows the data needed to calculate the molecular weight of the proteins
containing the EYFP marker.
Distance
(cm)
Mito
Golgi

14
12.
6

Actin

6.7

Mito

14

11.
6

Rf
0.8
8
0.6 0.
6
6
0.4
2
0.8
8

Molecular
Weight
12.75
34.15
69.44
12.75

41.77

Computer Analysis:
Aligning each of the four protein sequences against each other showed a
genetic imminence of 99% identity between the golgi and mitochondrial proteins,
86% between the actin and golgi as well as a 70% identity between the actin and
tubulin (table 3). This similarity though does not pass to molecular weight as the
sequences provided deviated weights. These weights, though, do not match those
found from the gel for mitochondria or tubulin but match nearly identically for the
golgi and actin proteins(table 4).
Table 3: The aligned protein sequences of each of the proteins and their identities.
Mit
o
Mito
Golgi

Golgi
238/239(9
9%)
x

Actin
Tubul
in

Actin
8/21(3
8%)
6/7(86
%)
x

Tubulin
6/14(43
%)
6/15(40
%)
7/10(70
%)
x

Table 4: Shows the comparison of the theoretical molecular weight from the net
exercise and from the gel calculations.

GFP
Mito
Golgi
Actin
Tubuli
n

Theoretical
pI/Mw:
5.82 /
52075.75
6.20 /
30765.09
6.21 /
36016.04
5.52 /
69438.08
5.19 /
77749.84

Theoretical MW
(KDa)

Calculated MW
(KDa)

52
30.8

12.75

36

34.15

69

69.44

77.7

12.75

41.77

EYFP Identification:
When the four sequences were aligned together a distinct sequence was found in
each of the four.
MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQSTVPRARDP

was found to be present near to the 3 end of each of the sequences and identifies
as the EYFP gene that each sequence shares(table 5). The EYFP sequence is 7.48
KDa compared to the 52 KDa of the GFP sequence (table 4 & 5).

Table 5: Shows the MW of each protein as well as the MW from web.expasy.org. The
tag was located along the 3 prime end of each of the proteins.
Protein
EYFP
Mitochond
ria
Golgi
Actin
Tubulin

MW Weight
(KDa)

12.75
34.15
69.44
12.75

MW-net-ex
(KDa)
7.5

MW-w/o EYFP
(KDa)
x

30.8
36
69
77.7

23.30
28.50
61.50
70.20

Tag
Location
x
3'
3'
3'
3'

Table 6: Observations of the Hepa-1 and A7 with and without nocodazole.

Hepa-1

Mito
Golgi

Hepa-1 +
nocodazole

A7

A7 + nocodazole

defined structure,
fiberous, many
highly
small
concentrated
concentrations
areas

large, lacking
structure, lots of
EYFP in
large, fiberous cytoplasm

small
concentrated
centers, sone
structure visible

condensed,
specific areas,
few, small,
intense areas

large structures
visible, intense
spots, ringlike
structures

EYFP in
cytoplasm, larger
area, weak
intensity

present

Actin

concentrated,
well defined
fibrous structure

well defined
structures, low
Tubulin intensity

more intense
than nonnocodazole but
intense, defined lots of EYFP in lots of EYFP in
fibrous structures the cytoplasm cytoplasm

well defined
structures, low
intensity

very
dissociated,
fibrous
structures
visible, not
intense

very dissociated,
fibrous structures
visible, not
intense

1)Each cell image showed a variety of similarities between the Hepa-1 and A7
organelles. The mitochondria showed well defined structures with many points of
intensity. The golgi showed small pockets of high intensity and a lack of intensity
elsewhere. The actin showed concentrated fibrous structures that had well defined
fibers. The tubulin had rather light wide structures that seem to be fibrous.
2)The A7 and the Hepa-1 cells even though from rat and mouse respectively,
differ in the fact that they are from muscle and liver respectively. The Hepa-1
mitochondrial proteins are much smaller than the A7 proteins and are more
condensed, likely because the muscle cells need larger mitochondria to provide
more energy than the liver cells. The golgi proteins are nearly identical in each and
show little variance. The actin show that the A7 actin are much less intense and
likely more flexible than the intense, well defined Hepa-1 actin. The tubulin, like the
actin, is well defined in the Hepa-1 cells and not so much in the A7 cells. This is also
suggestive of a stiff cytoskeleton in the liver, as it does not move, and a light,
flexible one in the muscle.
3)The nocodazole is used to prevent polymerization with cytoskeleton
microtubules. An intensity drop shows a lack of connection to the microtubules. In
the mitochondria of both animals the nocodazole caused a slight intensity loss but
overall the same structure. The golgi on the other hand vastly lost intensity in the
presence of nocodazole and became more intense in smaller pockets. The actin
structures gained some intensity as they are microtubule proteins and became

more defined. The Tubulin proteins showed little change with the addition of
nocodazole. The nocodazole reduces intensity in organelles not directly associated
with microtubules. This shows that all the organelles have at least some
microtubules. Those that compost the microtubules, the actin and tubulin, do not
diminish.

DiscussionGeneral1)The gel sandwich is placed in the orientation with the nitrocellulose

between the gel and the positive terminal so that the protein will travel into the
nitrocellulose membrane. If the orientation were flipped then the nitrocellulose
membrane would be blank and the proteins would travel in the other direction.
2)The blocking solution reduces the chances for the antibody to attach to site
other than the specific binding site on the protein. This prevents noise and clarifies
results.
3)The Hepa-1 mouse cells provide another perspective to the variation of
morphology between it and the A7 rat cells.
4)Consistent amounts of protein in each sample allows for a better
comparison of band intensities in the gel. This is to allow for identification of
multiple bands or bands that are very close together that can be compared to the
other lanes.

EYFP and Nocodazole:

1)Each of the organelles provided a different pattern in the EYFP. The
Mitochondria contain their own cytoskeletal network and only lose a little intensity
in the nocodazole while the golgi lose most of their intensity as they do not contain
large amounts of microtubules and mostly use them for transportation of other
proteins, as shown from the blots of high intensity. The Actin and tubulin are directly
part of the cytoskeleton and do not lose any intensity due to the nocodazole.
2)The blot shows the mitochondrial proteins are dark bands while the others
are light. This may be because the mitochondria are very large proteins while actin
and tubulin are smaller, tough tubulin is the same weight and mitochondrial
proteins and thus is more compacted.
3)The A7 cells showed less cytoskeleton and larger mitochondria than the
Hepa-1 cells as indicative of the types of cells, muscle and liver respectively. The

muscle cells need to be more flexible as to allow for large movements and the
mitochondria need to be large to allow for more evergy for the movements.
4)The nocodazole reduced the intensity in organelles where microtubules are
less populous. Both animals showed a reduction in intensity for the mitochondrial
proteins and a vast decrease in the golgi but a slight increase in both the actin and
tubulin.
5)The EYFP chimera appeared spread out in the surrounding areas of the
regions in only the A7 cells. This primarily occurred in the tubulin, as well as the
golgi and mitochondria in the prescience of nocodazole, and in the actin region
without the prescience of nocodazole. This could be due to the need for
microtubules to designate the location of structures in liver cells as transportation is
important to keep the liver, which performs a large amount of the bodys
metabolism, active.
Western Blot and Internet Calculations:
1)See table 4 and 5. The EYFP and GFP proteins share no amino acid
sequences together which is not unexpected as they are both relatively small
sequences. The EYFP is a functional protein but it is unknown if it was function
during this experiment as it was not tested for its chemiluminescence but it was
successful in the binding of EYFP antibody as the secondary antibody was able to be
identified in large amounts, as to rule out random pairing, and requires the EYFP
antibody to bind to the host.
2) The EYFP tag causes inactivity of some proteins as the fusion of the two
may disrupt the nature of one. EYFP is 27 KDa and thus is not a small protein. When
attaching to a smaller protein it may disrupt its secondary structure and denature it.
EYFP may also cover up or block the binding site of the other protein causing it to
be inhibited.
3)If the EYPF tubulin and actin were relatively half of the golgi and
mitochondrial proteins then the cell would likely be a more flexible cell that is likely
to be stretched and condensed more frequently than one with relatively equal levels
of the proteins. The levels of the EYFP chimeras would change based on the location
the EYFP was spliced into. If the EYFP hindered the protein it would likely be broken
down thus reducing the overall expression of the EYFP chimeras. Results similar to
the four Chimeric proteins mentioned may be found in other similar cell lines but
may also be found in very different cell lines. The variation of the secondary
structure of other proteins may alter the expression of EYFP as is primarily binds to
the 3 side of the protein.

ReferencesPurva Vats, Ji Yu, Lawrence Rothfield. The dynamic nature of the bacterial
cytoskeleton. Cellular and Molecular Life Sciences. October 2009. Volume 66. Issue
20. pp 3353-3362.
Richard Pollenz, Mary Kimble, Andy Cannons. Experiments in Cell Biology.2008. p5657.

L. Herrera-Estrella, M. De Block, E. Messens, J.-P. Hernalsteens, M. Van Montagu, J.

Schell. Chimeric Genes As Dominant Selectable Markers In Plant Cells. EMBO J.
1983; 2(6): 987995.

Roger Y. Tsien. The Green Fluorescent Protein. 1998.