It was concluded that he cells were not differentiated enough to begin with, the amount of cells used was not sufficient and the differentiation media did not have a neutral pH at the time of harvest. Introduction The purpose of the experiment was to differentiate CHECK cells into muscle cells and tests for presence of muscle protein specifics. The hypothesis was that proliferating CHECK cells could be induced to differentiate by removal of growth factors and differentiation would be characterized by expression of the muscle specific protein myosin light chain.
The rationale is that only differentiated muscle cells have the useless-specific protein myosin light chain (ML) thus ML can only be detected on differentiated cells. A gel electrophoresis separates the myosin light chain by size. Then a Western Blot using primary and secondary antibodies is used to bind the specific protein. An enzymatic reaction is then be produced to show light and allow the protein be captured on digital image. The cells used in the experiment were mouse CHECK cells, which are fibroblast-like cells that proliferate.
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Fibroblast cells start out as separate cells that look Jagged but are not fused together. In the residence of growth factors, the CHECK cells can be maintained in the undifferentiated state. They are also referred to as immovably, which is a muscle precursor cell. Moonbeams are determined cells that are committed to differentiating into muscle cells but can still proliferate. They only differentiate when a signal is given for them to exit the cell cycle. The growth factors necessary for proliferation are found in fetal bovine serum (BBS).
Replacing the BBS with 1-2% of horse serum will cause the cell cycle to arrest in GIG and initiate the differentiation process. Differentiated muscle ells will fuse together into multifaceted cells that look striated and long. The differentiated and undifferentiated muscle cells were lased and the protein was extracted and collected. The collected proteins are placed in well on an SD-Page gel and an electrophoresis is run. The myosin light chain protein should show up as a band corresponding to Qaeda in size. This process separates the proteins by their sizes.
The different proteins are then transferred over to a PDF membrane in exactly the conformation that they were in on the gel after the electrophoresis was run. A abbot polygonal primary antibody is used to detect the ML. Then a secondary goat, anti-rabbit antibody is used to detect the primary antibody. The secondary antibody also has an enzymatic site on it for a substrate to bind and cause an enzymatic reaction creating light. The overall reaction is HRS + alumina + hydrogen peroxide reacting to give off light. The light can then be detected with a digital images.
This means that any light signals captured will indicate that ML is present depending on the position on the membrane. The first step tot muscle cell determinations involves he inactivation of the cell cycle through depopulation’s of the Orb protein. Depopulation’s actually activates Orb protein, which then function to prevent the expression of genes required for the cells to enter the S phase of the cell cycle. In the second step, the proteins MOOD and Miff are activated. These proteins are transcription factors that activate the expression of genes required for muscle cell differentiation.
After growth factors are removed, the mystery express monogenic but can still be active in the cycle and are sensitive to patriotic cell death (Walsh and Perlman’s 1997). Once pop is induced, then the mystery irreversibly withdraw from the cell cycle and are less prone to optimism (Walsh and Perlman’s 1997). After the post-mitotic state is achieved, expression of miff and Mood is expressed. These proteins cause the perpetuation of monogenic and Miffs. Different members of the NEFF family such as MAFIA, B and D are up regulated and also miff is expressed (Yuan and Wool, 1996).
The last muscle regulatory factor to be activated in most muscle types is MRS. where it functions in the meteor or later in myofibril maturation and maintenance (Yuan and Wool, 1996). Finally the differentiated cells fuse into mottles. The regulatory protein Mood has a central role throughout the differentiation process of muscle cells. It initiates the monogenic differentiation pathway and that pathway temporally regulates the activity of Mood (Taproots, 2006). Mood is thus regulated by its own activity in a process called feed-forward mechanism.
For example, Miffed, which is activated by Mood in an earlier step, will cooperate with Mood in a later in the pathway to activate a subset of genes between 24-48 hours after induction (Taproots, 2006). This mechanism keeps Mood active throughout the entire pathway where it will bind directly to regulatory elements of genes expressed throughout the pathway (Taproots, 2006). By superimposing requirements for additional Mood-regulated factors at subsets of promoters, a temporal regulation is achieved (Taproots, 2006).
Methods and Materials The first part of the experiment was to split the CHECK immovably cells on a 100 mm plate and reseed them into four 60 mm plates. The media is first aspirated and 4 ml of PBS is used to wash away the media and aspirate. Then 2 ml of Trips was added ND set to incubate for 2-5 minutes to detach all of the cells from the plate surface. Next 3 ml of media was added to the plate and then the cell suspension was transferred to a 1 5 ml conical tube. After that, 500 LU was transferred to a centrifuge tube and the cell/ml concentration was determined under a hammertoes.
The concentration was used to determine the volume of suspension to add to each 60 mm plate. Plates 1 & 2 were added 150,000 cells or 0. 54 ml and plates 3 & 4 were added 300,000 cells or 1. 08 ml of suspension. After the four plates were incubated about 24 hours, the growth media of plates 2 ND 4 were changed to differentiation media (DMS). The media was changed on January 22 at 12:pm making the incubation time about 23 hours and 16 minutes. The media in plates 2 and 4 were warmed up to ICC before it was removed from the plates.
Both plates were then washed once with 3 ml of PBS and then 3 ml of DMS were added to each plate and then placed back in the incubator. After about five days, all four plates were taken out and washed with 2 ml of PBS and then aspirated. Then 0. 5 ml of PBS was added and the cells from each plate were scrapped and transferred, along wit PBS, too labeled micrometer tube and spun tort 2 minutes in a microelectronic. The PBS was then removed, leaving the cell pellet and the tubes were placed in a freezer. The cells are then lased and the proteins are extracted.
The lysine buffer contains TRIPS, EDIT, Nasal, glycerol, Triton X-OHIO, Noontide P-40 (NP-40), and protease inhibitors. The TRIPS maintains a neutral PH. EDIT is a chelating agent that binds to divalent actions to prevent protein degradation. Nasal helps else the nuclear and organelle membranes. Glycerol helps prevent protein degradation during freeze-thaw cycles. The use of NP-40 and Triton X-OHIO will help soluble hydrophobic proteins. The protease inhibitor prevents protein degradation from proteases released by the lissome.
About 5 pellet volumes of lysine buffer were added to each tube and then the tubes were incubated on ice for 15 minutes. Then the tubes were centrifuged for 10 minutes and the supernatant liquid was transferred to new tubes. The amount of protein in each tube was determined using the Commies dye- based Protein Assay (Bradford Assay). This allows an equal concentration of protein from each sample to be load into the wells later on when the gel electrophoresis is UN. The Commies dye is brownish/red in color but turns blue when mixed with proteins.
The blue color can be measured in a spectrophotometer at Mann. A standard curve was generated using known amounts of bovine serum albumin (BAS). Four microfilm tubes were set up and lull of Bradford reagent were added to each tube and lull of whole cell extract was added to each appropriate tube. Entire sample of four tubes were transferred to four wells on 96-well micrometer plate. The Absorption at Mann was measured for all of the samples and then recorded. In the next step, an SD polysaccharide gel electrophoresis (SD-PAGE) is setup.
In the SD-PAGE, proteins are denatured and their charge is neutralized before they are loaded into the gel so that they will be separated solely by their size. Boiling them for 3-5 minutes denatures the proteins and B-merchantable (BMW) is added to break disulfide bonds. Then sodium decoded sulfate (SD) coats the proteins with charged molecules to negate their charge. The gel is composed of two types, the lower resolving gel and the upper stacking gel. The resolving gel is made up of 2. 5 ml Creamily, 2. 5 ml TRIPS/SD buff, 5. 0 ml water, 100 LU of APS and 1 sulk of TEEMED.
This is poured into the gel plate holder and caster. While the gel polymerases for 15 minutes, a small amount of water-saturated butane is placed on top of the gel and then washed off after. The stacking gel is added afterwards on top of the resolving gel. The stacking is composed of 0. 5 ml Creamily, 1. 25 ml TRIPS/SD buffer, 3. 25 ml water, 50 LU of 10% APS, and 10 LU of TEEMED. A comb is placed between the plates to make the wells. The gels are then wrapped with moist paper towel and saran wrap and placed in the freezer until next lab.
Before electrophoresis is started, the comb is removed, exposing the wells and IX SD Running Buffer is added to fill the inner and outer reservoirs. A tube containing the protein molecular weight markers and the four sample tubes were boiled for 3 minutes then spun in the microfilm for 2 minutes. The molecular marker was loaded in the first lane. The lane two was load with 8. 8 LU of tube 1, lane three was loaded with 25 LU of tube 2, lane four was loaded with 18. 8 LU of tube 3, and lane five was loaded with 5. 5 LU of tube 4. The gel was run for 45 minutes at 180 volts.
Tater electrophoresis is done, the proteins were electro-eluted trot the gel and immobilizers onto a PDF membrane in their exact same pattern. Before transfer, the membrane is soaked in methanol and then transferred to buffer for 5-10 minutes. First piece of blotting paper was soaked in transfer buffer and placed on the bottom of the transfer apparatus. Then the equilibrated membrane was placed on the blotting paper and bubbles were rolled out. Next, only the resolving gel portion was removed and placed on top of the membrane and another pieced of blotting paper is placed on top of the gel.
A 20-volt current was run through the transfer apparatus for 20 minutes transferring the protein to the membrane. Finally the membrane was transferred to a solution of IX TABS with 5% milk and shook for 30 minutes. The IX TABS and 5% milk is a blocking buffer used to reduce nonspecific bind of the antibody. It was then washed with 2-3 changes of IX TABS and incubated overnight with the primary antibody. The primary antibody was diluted 1:500 in IX TABS + 5% milk. The membrane was then wash 3 times for 2 minutes each with about 10 ml IX TABS and then incubated with the secondary antibody for 30 minutes while shaking.
The secondary antibody was diluted 1 in IX TABS. The membrane was then washed again 3 times for 2 minutes each with IX TABS and then placed in a 1:1 dilution of Luminous and H2O for 2 minutes. The membrane was then placed on a digital images and an image was captured. Results The CHECK proliferating cells using at the beginning of the experiment are shown in Figure 1: Proliferating CHECK (MM). As the Figure 1 shows, these cells have a Jagged appearance and are separated by adjacent cells.
The number of cells counted in the top left quadrant of the hammertoes was 26, top right was 28, bottom left was 29, and bottom right was 28. The average of these was 27. 75 and the calculated concentration was 277,500 cell/ml. These results can be seen in Figure 2: Hammertoes count of Proliferating CHECK. The differentiating media was added to plates 1 & 2 on January 22 at 12:16 pm. The cells on all four plates were harvested on January 27 at around 9:00 am so the incubation period in DMS was about 116 hours and 44 minutes.
Before harvesting, the CHECK in the differentiating media were observed under the microscope. The CHECK in DMS became fused together becoming multifaceted and looked very long and striated. This can be seen in Figure 3: Differentiated CHECK at errs. At the time of harvesting the cells, the differentiating Edie for plates 2 & 4 also showed a yellow color. The whole cell extract turned from a white solution to light blue during the Commies dye-based Protein Assay. The volume (LU) of sample needed to load 40 GU, 35 GU, 20 GU, and 15 GU of that sample were determined for samples 1-4.
The volumes loaded were for guy of sample. This can be seen in Table 1 :Volumes of Sample to Load. Whole cell extracts from undifferentiated and differentiated CHECK cells were analyzed for expression of myosin light chain using Western Blot. The results showed that ML was present in the differentiated cells of sample 2 & 4 as a ere dark band in the digital image. The undifferentiated samples 1 & 3 showed very light bands. The band for sample 2 showed up much darker than sample 4, which had a cell density of 200,000.
The prediction was that only the differentiated samples would show a band at Qaeda where myosin light chain is suppose to show up and undifferentiated sample would show no band. The goal was to get the CHECK to successfully differentiate and show the ML band in the Western Blot but if the bands did not show then the goal was to understand what went wrong during the experiment for the bands to not show up. The results in the experiment did not support the hypothesis. The digital image did not show any bands corresponding to myosin light chain for the differentiated cells.
This was due to the fact the CHECK cells did not differentiate enough after incubation in differentiation media. At the time of harvesting, the cells in differentiation media did not show a good level of differentiation. There was a certain degree of differentiation but it was very low compared to the differentiated CHECK cells shown in Figure 3. Another reason was the lack of cells used. The amount used ere 150,000 cells for samples 1 & 2 and 300,000 for samples 3 & 4. Using more cells could have increased the amount of protein extracted later on in the experiment.
Since the amount of differentiated cells were already low, using a higher number of cells would have proved more beneficial because it would have given a higher yield extracted protein. Also plates 2 & 4 had yellow differentiation media at the time of harvesting the cells. A yellow media meant that it was acidic and and a pH tot about 6. 0. This was one possible reason why the cells did not differentiate very well and thus not much protein was extracted. There are several things that could be fixed had this experiment been repeated.
The amount of cells used for each plate would have to be increased to ensure a better chance of extracting protein. Doubling or tripling the amount of cells used could be done. The cells in the differentiation media would have to be monitored more closely to ensure that the environment is kept at optimal condition. In the actual experiment, the differentiation media was yellow when the cells were ready to harvest, which was a bad because the pH was acidic and not neutral. Closer monitoring of the medias could have prevented this.