Analysis of Chimeric Proteins by Fluorescence Microscopy and Western BlottingBy Pratik B. PatelGroupmates: Brandon Pierce, Kristine Geer, Christian WardPCB 3023L Sec # 006T.A: Kamisha Woolery4/14/2015

IntroductionThe cytoskeleton of complex eukaryotic cells consistents of three primary types of protein filaments actin microfilaments, which are about 7 to 8 nm in diameter and made of actin protien, microtubules hollow rod like filaments that are 25 nm in diameter made of alpha and beta tubulin proteins, and intermediate filaments which are very diverse and vary in specific cells (Reece, et al., 2010). The function of the Cytoskeleton to the eukaryotic cell is to provide support anchorage to other cells and surfaces. It also functions in cellular motion and communication with other cells (Reece, et al., 2010). This experiment aims to use chimeric proteins to study the importance of cytoskeletal proteins in the location and configuration of crucial organelles such as mitochondria and Golgi apparatus. Chimeric proteins are proteins which are tagged with an abnormal or purposefully modified polypeptide chain (Pollenz, Kimble, & Cannons, 2007). They are created using chimeras which are modified DNA or mRNA genes that encode the modified chimeric protein (Datskevich & Gusev, 2014). Biologically engineering chimeras requires taking a transplantable DNA fragment such as a circular DNA plasmid and adding in a specific sequence of the gene that is desired for expression (Ma, et al., 2012). The expression of the added gene is concurrent with the genes in the plasmid once a host cell takes up the plasmid it begins to express the genes in the modified plasmid with its own genome (Reece, et al., 2010). The specific chimeric protein induced modification in this experiment was toward cystoskeletal proteins, particularly actin and tubulin, which also surround the Golgi apparatus and mitochondria. The chimeric protein tag used in this experiment is a variant of the green fluorescent protein. Green fluorescent protein (GFP) is a protein that is responsible to fluorescent light visible in some marine organisms (Remington, 2011). It is a tool widely used in experiments related to the creation of chimeric proteins that glow and give off light upon stimulation to electromagnetic radiation so that static cell structures as well as dynamic processes can be visualized in the cell (Pollenz, Kimble, & Cannons, 2007). Enhanced yellow fluorescent protein (EYFP) is a variant of GFP that is used in this experiment and serves similar functions as the original GFP. The specific EYFP used in this experiment serves to either be targeted to Golgi apparatus or the mitochondria and other cystoskeletal protein fibers and it is to be visualized under florescent microscopy. This variant is caused by a shift in 6 amino acids substituent which shift the emission spectrum of the protein from the green to yellow wavelength (Pollenz, Kimble, & Cannons, 2007). The goals of this experiment are to find the localization of cytoskeletal tubulin and actin proteins in the cells near the Golgi apparatus and mitochondria, calculate the fluorescent chimeric proteins molecular weight along with the actual non-chimeric proteins (without the protein tag) weight and in what way is the EYFP fluorescent protein tag attached to the cytoskeleton proteins targets. Cytoskeleton proteins are important for the specific positioning and function of all the organelles in the cell. Another important goal of this experiment is to determine how the distribution of cytoskeleton proteins affects the position and function of the organelles in the cell. It is predicted that the microtubules are highly associated with the functioning of the Golgi complex and the mitochondria. Therefore, the addition of de-polymerizing agent like nocodazole in the cell would cause a more varied distribution of Golgi complexes and mitochondria throughout the cell (Pollenz, Kimble, & Cannons, 2007). Methods & MaterialsDeterminination of the Amount of protein in Each HomogenateHepa-1 cells, hepatoma cells differentiated via mouse liver tumor cells, were injected with enhanced yellow fluorescent proteins expression plasmids and were dissolved and homogenized in Laemmli buffer (0.125 M Tris HCl, set to a pH of 6.8, 20% glycerol, 4% SDS, and 10% -Mercaptoethanol). Aliquots of the protein samples were made with a concentration of 1g/L and were all designated as GOL for Golgi, TUB for tubulin, ACT for actin, MIT for mitochondria and HEP for non tranfected Hepa-1 cells. SDS PAGE Gel Electrophoresis An SDS PAGE gel was run for each of the protein aliquots on a 10% polyacrylamide SDS-PAGE gel using 1X Gel running buffer (25 mM Tris 192 mM glycine, 0.1% sodium dodecyl sulfate:SDS) for one hour (Bio Rad Laboratories Inc, 2014). About 20g of protein were loaded in each lane and one lane was loaded standard molecular weight marker. The markers were loaded as according to the following scheme shown in Table 1.Table 1: Gel Loading Orientation for SDS PAGE Gel ElectrophoresisLane123456

SampleMW MarkerGOLTUBACTMITHEP

Volume (L)202020202020

Table 1: This table shows all of the samples that were loaded onto the poly-acylamide gel and the volume each well was filled with each sample. MW marker was placed in well 1 stands for the molecular weight standard used and the different protein and the non-modified HEP The Gel was run for 1 hour at 200 volts until the dye front had reached the bottom of gel. Once complete the gel was removed from its case and placed in nitrocellulose transfer buffer containing: 4% Tris-gylcine transfer buffer 25x, 20% methanol and water (Bio Rad Laboratories Inc, 2014). Transfer of the Protein to Nitrocellulose (Western Blot) A transfer clamp, two pipette tip box lids (one small one large), 1 piece of nitrocellulose 2 Scotchbrite sponge pads and 4 pieces of Whatmans 3MM blotting paper were gathered. The nitrocellulose was submerged in nano-pure water. On the transfer clamp colored black indicating the negatively charged side a sponge was placed on the black side, after which two pieces of blotting paper and then the protein still on the gel were placed on the clamp. The nitrocellulose membrane was then placed on the gel facing the protein. Two more pieces of blotting paper was added on top of the membrane and then another sponge. The positive white portion was then sealed on top of the sponge and the clamp was sealed. Before each layer was applied in the clamp it was dampened in transfer buffer. Once the clamp and the western blot transfer layer was made it was ran in a transfer apparatus for 1 hour at 100 mA. Processing of the Western BlotThe blot on the nitrocellulose was taken from the clamp after it had finished running. Ponceau S dye was added to the nitrocellulose blot and was soaked in the dye for a few seconds. It was then taken our and rinsed with water. Once the dye is washed off the nitrocellulose blot was placed in 5% dry mild solution for 30 min. After this the blot was washed with phosphate buffered saline (PBS) buffer and then stored. Staining the Western BlotThe blot was placed face down into a tray containing blocking solution made from TBST (Tri buffered Saline: 50 mM Tris, 150 mM NaCl and0.05% Tween) with 5% dry milk and incubated for 25 min. The blot was removed from its wrap and then placed fully into 15mL of TBS for a few min. The TBS solution was then filtered out from the blot and a 1:250 primary antibody solution was poured onto the blot so that it was fully submerged. It was rocked back and forth. It was then wrapped with saran and then put on an automated rocker for 45 min. The antibody solution was then rinsed and about 10 mL of TBST wash solution (0.1 M NaCl, 0.5 Tris, pH 7.4 0.03% Tween-20) was used to rinse the blot. And about 20mL of it was added to submerge the blot and incubated for 8 min. The blot was then dumped out from the blot and washed again two more times. The last TBST buffer was dumped and then a 1:1000 antibody solution was added to the blot and incubated for 35 min. Again this antibody solution was dumped and 10 mL TBS wash buffer was used to wash the blot. The blot was again soaked in 20mL of buffer for 8 min and repeatedly rinsed for 8 min. After the second wash, the TBST was poured out and the blot was positioned face up. Eight milliliters BCIP/NBT blot solution was added to the blot and rocked back and forth for uniform spreading. The blot was observed for 10min until bands became visible on the blots. After these bands were uniquely visible and intense they were rinsed with distilled water for 3 min and then submerged in water. The blot paper was then taken out and placed on a sheet of blotting paper. The molecular weight marker and the EYFP to the dye front had measurements made in centimeters and Rf values calculated. Table 2 below shows all the steps of the Western blot that were done in this experiment along with proper volumes of each of the substances used and the intervals of time between each step.

Table 2: Outline of All the Steps for a Western BlotStepSolutionVolume used (mL)Time interval (min)

BlockingBlocking solution1525

Primary AntibodyBlocking solution545

PrewashTBST10Rinse

WashTBST208

WashTBST208

WashTBST208

Secondary AntibodyBlocking solution1035

PrewashTBST10Rinse

WashTBST208

WashTBST208

WashTBST208

WashTBST10Rinse

WashTBST10Hold

DevelopmentNBT/BCIP810 min

Table 2: This table shows all the steps taken to complete the western blot done in this experiment. It includes the each step with the appropriate solution, solution volume and wait times between each step. Graph and Determining Difference in Molecular WeightMolecular weight for the EYFP protein was determined by using the standard weight marker and the Rf values for each of the marks to create a standard curve for the proteins weight. This standard curve was used to determine the weight of the EYFP protein by using the semi-log equation of the molecular weight marker and the Rf of the EYFP protein band.Theoretically Determining Molecular Weight of EYFP Chimeric Protein Proteomic sequencing and comparing program was used to determine the molecular weights of the EYFP protein and the chimeric protein so that the proteins molecular eight without the EYFP tag could be determined. The website and program used to transcribe and translate the DNA embedded with EYFP expression was called http://www.expasy.org/ . transcription. Once this was translated this amino acid sequence had its molecular weight found by using the ProtParam program available at this website. The molecular weights for chimeric proteins in which EYFP was bound and tagged in this experiment were also found using their amino acid sequences. This included EYFP tagged chimeric proteins that were ran in the SDS PAGE gel: MIT (mitochonrida), GOL, (Golgi), ACT (actin), TUB (tubulin). Differences between molecular weights was calculated to find the molecular weight of the proteins without the tag under normal cell conditions. Protein AlignmentProtein alignments were done using the protein blast program which rigorously analyses differences in the protein amino acid sequence indicating differences in the proteins. The website used is http://www.ncbi.nlm.nih.gov/. The wild type EYFP sequence was compared with chimeric protein sequences for MIT (mitochonrida), GOL, (Golgi), ACT (actin), TUB (tubulin). The number of differences in the sequence was determined from these protein blast comparisons as well as which end the tag is attached to (N terminus or C terminus).

ResultsDifferences of Microtubule Orientation in Nocodazole and Non- Nocodazole Treated CellsDifferences in the Nocodazole and non treated cells were observed for each tagged protein of Helpa-1 and A7 cell lines, and the following observations were recorded in Table 3 below on the next page.

Table 3: EYFP Fluorescent Microscopic Observations of Hepa-1 and A7 CellsCell types and Protein Clustering

TaggedChimeric proteinHepa-1Hepa-1 w/ NocodazoleA7A7 w/ Nocodazole

MIT Uniform expression throughout the cell clustered around mitochondria More protein aggregation and clustering throughout the cells mitochondriaAppears slightly less clustered throughout the cells organelles Appears only slightly more clustered throughout the cell

GOLMuch more clustered and aggregated expression around GolgiLess aggregation and more visibly smaller clusters dispersed throughout the cells GolgiClustered in the cytosolMore spread out through the cytosol

ACTSpread uniformly throughout the cellsSpread uniformly throughout the cellsZig-zag fibrous uniform spread of proteinZig-zag less fibrous but still uniform spread of protein

TUBSpread uniformly throughout the cellsSpread uniformly throughout the cellsSpread uniformly throughout the cellsSpread uniformly throughout the cells

Table 3: This table shows the observations for hepa-1 and a7 cell lines that had various cytoskeletal proteins tagged with EYFP and how the distribution of tagged proteins differed with the addition of an anti polymerizing agent. Descriptions were given in terms of the distribution of colored proteins and the whether they were clustered or uniform and how clustering or uniformity varied from Nocodazole treatment. Differences between Treated and Untreated Hepa-1 and A7 Cells. The difference between the two cell lines A7 and Hepa-1 are the cytoplamic apace the hepa-a cells have a larger cytosol space and tend to have more prominent branching. The uniformity of the cytoskeleton proteins of each cell are similar except when untreated with nocodalzole the proteins in the hepa-1 cell appear more clustered. However, upon treatment the proteins throughout the cytosol respond and become distributed in a similar fashion. Initial untreated proteins in the A7 cells are more condensed and fibrous while the cells proteins in hepa-1 are more dispersed and clumpy throughout the hepa-1 cells. The observations in Table 3 show that the distribution of TUB and ACT proteins did not change much from one another and the initial untreated tagged cells showed no real difference when treated with noncodazole. However, the distribution of cytoskeleton protein tagged and aggregated around the mitochondria and Golgi showed more differences when treated with noncodazole for example comparing Golgi tagged proteins in both hepa-1 and 7 cells. Western Blot and Experimental Molecular Mass of EYFPFigure 1 below shows the western blot done to determine the molecular mass of the EYFP. Figure 2 shows the standard curve for the molecular weight marker on the western blot of Figure 1.

Figure 1: Western Blot of EYFP

Figure 1: This image shows the actual gel western blot that was produced of determining EYFP molecular weight. Labeled on the left had side is the molecular weight markers and their corresponding weights. Labeled at the top and bottom are the places the ends of the blot were taken as so that Rf values for the proteins could be calculated. The total distance between ends was 13.85 cm. Also labeled are four prominent blotted dye fronts that labeled from A to D. The running conditions for this blot were 100mA for 1 hour within the transfer clamp detailed in the Methods & Materials section. The western blot in Figure 1 shows four fragments from A to D that could represent the weight of the EYFP chimera.

Figure 2: Western Blot Standard Curve of the Molecular Weight Marker

Figure 2: This standard curve shows the molecular weights in log scale of the molecular weight standard shown in Figure 2 on the y-axis. The x-axis shows the Rf (relative mobility) of those six bands that are seen after running and staining the blot ransfer. Also presented in this figure is the R2 of the exponential-log trend line equation which was used to determine the molecular weight of protein fragments blots A, B, C and D. The standard curve in Figure 2 presents a standard curve semi-log equation that is used to determine the mass of the EYFP chimera on the western blot. Also shown is a trend line and a R2 value greater than 0.98. Table 4 below shows the calculated protein masses from the standard curve and the Rf values of A, B, C, and D from Figure 1.

Table 4: EYFP Western Blot Molecular Weight From Dye Blots (next page)Blot dye frontsRfMolecular weight (Daltons)

A0.79422416249.7

B0.83032514052

C0.87364611803

D0.72924221107.24

Actual WeightN/A26914.3

Table 4: This table shows the Rf values for each of the each of the dye blots seen in Figure1 as well as their molecular weights determined from the trend-line equation in Figure 2. Also shown for comparison is the actual weight of EYFP protein determined by the sequencing and Expacy ProtParm program. The closest value of to the actual proteins molecular weight was dye blot D which was 21107.24 Daltons and is only 5807 Daltons difference.

Table 5: EYFP theoretical EYFP ChimeraEYFP molecular weight (Da)EYFP tagged chimera molecular weight (Da)Untagged protein weight (Da)Tag location (COOH or NH2FunctionalityNumber of Amino acid differences

MIT26914.330765.03850.7COOHNon functional7

GOL26914.336016.09101.7COOHNon Functional6

ACT26914.369438.042523.7NH2Functional 6

TUB26914.377749.850835.5NH2Functional6

Table 5: This table shows the molecular weight of EYFP protein molecular weight. The tagged EYFP molecular weights are also shown. The proteins without the EYFP tag molecular weights are presented and the location in which the EYFP tag is attached into the protein as well as the number of amino acid differences It also presents whether the proteins are functional. The details of these data were obtained through Expacy and the protein blast program by the national institute of health. The molecular weights of the chimera for untagged TUB and ACT are the highest as indicated in Table 5. Both of these proteins also have the EYFP tagged on their N-terminal end. The MIT and GOL protein chimeras have the tags at the C terminus end. There were more differences in the amino acid sequence for the MIT and TUB protein than the GOL and ACT(6v.s. 5).

Table 6: Differences in the Amino Acid Sequences of EYFP and Chimeric EYFP Proteins Query and SubjectSequences

Query: MITsbjct: EYFP wild type

Query: GOLsbjct: EYFP wild type

Query: ACTsbjct: EYFP wild type

Query: TUBsbjct: EYFP wild type

Table 6: This table shows the actual matching sequences between the wild type EYFP protein and the tagged chimeric proteins used in this experiment. The mismatched or gap sequences are highlighted and their positions are also shown. The subject (subject) was taken to be the wild type EYFP protein sequence while all of the queries were the chimeric EYFP tagged proteins.

Discussion Hepa 1, A7 Cells and Fluorescent MicroscopyThe pattern of expression for MIT tagged proteins in both Hepa-1 and A7 cells was largely around areas of the mitochondria. Both none treated cells showed fairly regular clustering of proteins around the ATP producing organelle. Expression in GOL was oriented near the Golgi complex further from the cells nucleus and closer toward its membrane. The expression in both cell types showed much clustering of these proteins throughout the cytosol. Expression for TUB and ACT were very similar in both cell types both showed uniform spread of the EYFP fluorescent proteins throughout the cell in the cytosol but, the A7 non treated cells seemed to have more fibrous actin filaments than the Hepa-1 cells. This could likely be due to the fact that the Hepa-1 cells are cancerous liver cells and the A7 are muscle.The pattern of protein clustering itself is different in both cell types in terms of the exact location of and spread. For instance, the MIT cells in A7 were less clustered and spread more uniformly through the cell. This is likely because here mitochondria in muscle cells such as A7 cells need to be more spread out for the rapid breakdown of ATP. The Golgi protein clustering was about the same for both cells although the Hepa-1 cells seemed to have a few more clusters than A7 cell type. This is likely because the hepa-1 cells are were cancerous successors of healthy liver cells. Nonocodazole treatment essentially served to break down polymeric cytoskeletal proteins and so that the differences in expression could be elucidated from the results. The results of treatment form the de-polymerizer show less expression of the protein in concentrated areas and expression in more areas of the cell. For example, Nonocodazole dispersed the proteins and made them more uniformly spread out throughout the cell due possibly to the breakdown of the polymer in which the EYFP tags are attached to. The response was roughly the same for both cell types although the effect for MIT in Hepa-1 cells appeared more pronounced than the A7 cell type. This could be because there is already a wide spread of mitochondria in the A7 cells so have more broken down parts is difficult to distinguish from whole parts. Overall, it appeared that the microtubule proteins did have an impact on the organization and functioning of cellular organelles due to the noticeable clustering of the tagged EYFP proteins in the observed cells. The MIT had more clustering in the non chemical treatment with nonocodazole group because it likely served a vital role in supporting the cells organelle. Upon treatment the proteins became more dispersed as expected. This was observed in both cell types however more so in the hepa-1 cell type. In the GOL chimeras the same was observed more dispersed expression of EYFP proteins in the cytosol upon treatment. This is again attributed to the separation of the protein filaments by the de-polymerizer. Western Blot Analysis The transfer of proteins from the SDA PAGE gel to the nitrocellulose film depended on the proper orientation of the Gel and the film in the transfer clamp sandwich. The sandwich transfer clamp for the western blot was arraigned so that the proteins with a negative electric charge would travel to the positive side from the negative side onto the nitrocellulose film. If the arraignment was reversed the nitrocellulose would not be blotted correctly and the western blot would have no proteins having shown up on the film even after the lengthy staining process. The blocking solutions had an important role in the western blot process as well. Blocking solution is used to stick the antibodies to the protein so that they are able to collect dye and be able to stain effectively so that the protein transfer can be resolved. Likewise it is equally important to load all the samples with each protein because this could also lead to difficulty interpreting results. Some of the bands on the western blot could come out fainter or more intense depending on the relative amount of protein that the well was loaded with. This would lead to difficulty in interpretation of the results or may lead to an inaccurate results. Another method that was employed to prevent inaccurate results was the addition of a Hepa-1 as a control as shown in Table 1. This was used so to make sure that no extra contaminant was causing none tagged proteins to show up present on the nitrocellulose gel. The absence of this from the western blot indicates that only tagged protein was able to be transferred onto the film. The intensity in the color of the western blot as compared with that of the fluorescent dye tags seemed to be unequal. The intensity of the green protein in the fluorescent pictures suggests greater expression of the proteins and much more aggregation near organelles and throughout the cytoplasm. This difference suggest that some of the protein was not all transferred successfully o the nitrocellulose film therefore it lacks the same intensity seen the fluorescent micrographs. It is also possible some of the chemicals during the washing and staining process of the western blot could have un-intentional removed some of the protein and thus produced a stain less intense than the proteins that are expressed in the micrographs. Theoretical EYFP and Chimera Calculations and Western BlotThe tag that is added to a fully functional protein cans inhibit proper protein function because its peptide chains no longer have the same set of unique amino acids. This in term changes some of the secondary structures of the protein and causes the protein to bind or become unresponsive to what it should bind to (Remington, 2011). If there were a difference found between of the amount of protein expressed in the Hepa-1 and A7 say more actin in A7 than hepa-1 cell lines. Than with regard to actin filaments a reasonable explanation can be given to help explain this phenomenon. It is because actin filaments are important in the usage of ATP breakdown are one of the essential proteins necessary for contraction of muscle. Therefore it would be obvious that A7 muscle cells have more actin which is required for their specific function. This would be expected in of course other different types of cells with varied functioning.In conclusion the results of this experiment suggest that the cell cytoskeleton is indeed important for the functioning and positioning of cellular organelles and intra-cellular traffic. It was also found out that the western blot of the EYFP chimera proteins were inconsistency with the theoretically calculated molecular weight. This is likely due in part to the transferring process being done incorrectly or some random error associated with the experimental set up. It is possible in further experimentation to redo the western blot with more concentrated protein concentrations to ensure reasonable transfer of protein to the western blot film. The masses for the non tagged proteins were found out by successfully using proteomic analysis and comparison programs.

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