Collection of tissue from mouse
C57/BL/6 mice were humanely sacrificed by asphyxiation followed by exsanguination. Heart, lung, diaphragm and kidney tissues were excised and were immediately transferred to liquid nitrogen (-70°C) and afterwards onto dry ice (-78.5°C) to prevent proteases from breaking down the protein of interest which in this experiment was Akt. Proteases break down proteins by hydrolyzing peptide bonds that link amino acids in the proteins. The very cold temperatures used in processing the samples in the experiment reduced protease activity and preserved the integrity of the Akt, which enabled us to study its expression in the tissues.
Homogenization of the excised tissues
The tissues were homogenized with the aid of a tissue homogenizer (Tissuemizer). The entire process was maintained on ice in order to slow down protease activity. Also, cOmplete Minitab (Cat# 1836153, Roche Diagnostics Corporation, Indianapolis, IN) was added to the lysis buffer to inhibit protease activity. The homogenizer dispersed the tissues into very small pieces in order for the ice cold lysis buffer (containing 1.0% NP40, 10% Glycerol, 20mMTrizma, 137mMNaCl, 1mM MgCl2.6H2O, 1mMCaCl2 dihydrate) to efficiently lyse the cells to release the proteins out of the cells into the lysate. The debris formed sediment after centrifugation at 14000rpm at 4°C while the supernatants containing the Akt were saved for Western analysis.
Determination of total protein concentration
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Total protein concentration of the lysates was calculated to determine the amount of protein extract to be loaded on the PAGE gel. By so doing, we were able to load equal amounts of the various lysates in order to compare their band intensities on the Western blot. To do this, we employed the use of the BIO-RAD protein assay reagent (Cat# 500-0006, BIO-RAD, Hercules, CA). This works by the Bradford assay principle (1) which is based on the color change of Coomassie brilliant blue dye in response to various concentrations of proteins. In an acidic medium, the dye binds to basic and aromatic amino acid residues. This causes the reddish brown dye to change to a blue color whose absorbance can be measured with reference to the absorbance and concentrations of bovine gamma globulin protein standards (Cat# 500-0208, Bio-Rad). The absorbance was measured at 595nm due to the fact that the difference between the reddish/brown and the blue color of the dye is best observed at 595nm. The various absorbance values for the standard protein samples that were run alongside the samples were used to construct a standard curve. The equation of this standard curve was used to calculate the concentration of the protein extracts.
Polyacrylamide gel electrophoresis (PAGE)
This uses the principle that protein can migrate through a gel matrix under the influence of an electric field based on size and charge. The samples to be run on the 4-12% NuPAGE gel (Cat# NP0321BOX, Invitrogen, Carlsbad, CA) were first boiled at 90°C for 5mins in loading buffer composed of an NP LDS sample buffer (Cat# NP0007, Invitrogen) and reducing agent (Cat# NP0009, Invitrogen). This gel concentration was selected in order to achieve at least 70% migration of the protein of interest through the gel matrix from the point of origin. The NP sample reducing agent (Cat #NP0009, Invitrogen) contained DTT (dithiothreitol) which reduced any disulfide bridges that held together the tertiary structure the proteins in the lysate. The proteins were made to assume an overall negative charge by the treating the lysates with LDS sample buffer (Cat#NP0007) containing an anionic detergent LDS (lithium dodecyl sulfate). The LDS bound strongly to and denatured the tertiary structure of the proteins while applying an overall negative charge to the proteins in the extract. Thus, the proteins in the lysate were made to open up into rod-like shaped structures with a series of the negatively charged LDS molecules along their polypeptide chains. Heating the samples to 90°C helped to further break any remaining disulfide linkages in the tertiary structure of the proteins. We did this because the LDS and the reducing agent may not have completely cleaved all the disulfide bonds. A hole was poked in the cover of the tube while the heating was done to prevent the reaction mixture from over boiling. The sample buffer also contained a tracking dye which helped us to monitor the electrophoretic run. SeeBlue® Plus2 pre-stained standard (Cat# LC5925, Invitrogen) was run alongside the various tissue protein extracts. This standard was composed of calibration proteins of known molecular weights, including myosin 188kDa, phosphorylase 98kDa, BSA 62kDa, glutamic dehydrogenase 49kDa, alcohol dehydrogenase 38kDa, carbonic anhydrase 28kDa, myoglobin 17kDa, lysozyme 14kDa, aprotinin 6kDa and inulin β chain 3kDa. We compared these bands with that which we obtained for Akt 60kDa to ensure that the band fit the molecular weight of Akt. Because the standard was pre-stained we were also able to know the correct orientation of the blot for imaging after transferring the proteins by electroblotting from the gel on to the membrane.
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In this experiment, we used electroblotting to transfer the separated proteins in the gel on to a membrane with high protein binding capacity such as nitrocellulose or PVDF (polyvinylidene fluoride). An electric current was used to pull the proteins from the gel on to a PVDF membrane. PVDF has a high protein binding capacity, strong hydrophobicity and solvent resistance which made it the membrane of choice for the experiment. The PVDF membrane (Cat# LC2002, Invitrogen) used for this experiment had a pore size of 0.2µm.This allowed the proteins to migrate from within the gel on to membrane with minimal damage to the protein bands and without any distortion in how they were arranged on the gel. The blot was labeled with a pencil since blue ink would bleed into the blot and caused interference during the imaging process. This is due to the fact that the LI-COR Odyssey® infrared imaging system detects blue color as fluorescence and this interferes with the actual fluorescence produced by the fluorescently- tagged antibodies bound to the blot.
After transferring the proteins on to the PVDF membrane we were able to probe the blot for the protein of interest Akt using a primary antibody, mouse anti-Akt (Cat #BD610861, Becton Dickinson, Franklin Lakes, NJ) antibody which specifically binds to Akt1. Before this, we treated the membrane with Odyssey blocking buffer (Cat# 927-40100, LI-COR, Lincoln, NE) to reduce any background interference and non-specific binding of the primary antibody. The anti-mouse primary antibody was raised in mouse and was designed to recognize the Akt1 isoform, irrespective of the state of the Akt, whether it is in its phosphorylated active form or in its unphosphorylated inactive form. This primary antibody was used to treat the blot at a dilution of 1:1000 and this was incubated over three days since a slow but specific binding is known to yield much better results. The blot was washed in 1X PBS (phosphate buffered saline) (Cat#11666789001, Roche Diagnostics Corporation) containing 0.1% Tween-20 (Cat# P5927, Sigma-Aldrich, St Louise, MO) to remove any anti-Akt which did not bind to the Akt protein on the blot. The secondary antibody (Cat# 610-132-121, Rockland Immunochemicals Inc, Gilbertsville, PA) was raised in goat against mouse antibody. The secondary antibody was conjugated to IRDye®800, an infrared dye which has been optimized for use on the LI-COR Odyssey® imaging system. During the treatment with the secondary antibody the blot was protected from light to preserve the fluorescence of the secondary antibody-IRDye®800 conjugate. Thus, the secondary goat anti-mouse antibody (Cat# 610-132-121, Rockland Immunochemicals Inc) bonded to the mouse anti-Akt primary antibody (Cat #BD610861, Becton Dickinson) that was in turn bound to Akt on the PVDF membrane. The blot after treatment with the secondary antibody was washed again with 1X PBS (Cat#11666789001, Roche Diagnostics Corporation) containing 0.1% Tween (Cat# P5927, Sigma-Aldrich) to remove any unbound secondary antibody. This helped to avoid the entire blot from fluorescing during imaging. Thus, only the areas on the blot that the secondary body was bound to the primary would show fluorescence.
Imaging the Immunoblot
The LI-COR Odyssey® infrared imaging system was used to specifically detect the presence of the protein on the membrane by detecting the florescence of an IRDye® 800-secondary antibody conjugate using an infrared laser. When visible light is used to image, it produces high background and autofluorescence. For this reason, the use of an infrared laser is recommended because it reduces the occurrence of both autofluorescence and light scatter which produce background. On the Odyssey imager there are two infrared detection channels that detect fluorescence at 700nm and 800nm with the incorporation of optical filtering. The 100nm difference between these two detection channels ensures that each detector measures only fluorescence emitted from one infrared dye. The IRDye®800 conjugated to the secondary antibody used for this experiment is an infrared fluorophore which produces fluorescence at an excitation wavelength of 780nm. Thus, when the infrared laser is incident on the IRDye-secondary antibody conjugate on the blot it caused an excitation in the dye which resulted in a spectral shift causing the dye to emit light at a longer wavelength (780nm) which was detected by the 800 channel infrared detector.
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In this experiment, we examined the expression of Akt in the heart, lung, diaphragm and kidney of a mouse to determine if the level of expression of Akt was related to the role that the protein is thought to play in these organs. The concentration of total protein in the tissue lysates from the heart, lung, diaphragm and kidneys as well as the standards were used to calculate the concentration of the total protein in each of the tissue lysates. The correlation coefficient for the standard curve generated was 0.987 which meant that the standard curve could be used to extrapolate the concentration of the lysates (Table 1). Thus, equal amounts (50µg) from each of the mouse tissue lysates were loaded on to the acrylamide gel for electrophoresis.
After running the gel and imaging the blot on the LI-COR Odyssey imaging system, well separated ladder-like bands of the See Blue® Plus 2 pre-stained molecular weight standard (Cat # LC5925, Invitrogen) were observed. In each of the lanes of the lysates from the heart, lung, and diaphragm tissues there was a visible single band which corresponded to the 60kDa molecular weight of endogenous Akt (Figure 1). The western blot depicted the expression of Akt in the heart, lung and diaphragm but the expression of the protein in the kidney was not apparent in the image of the blot (Figure 1). The absence of a clear Akt60kDa band in the lane of the kidney lysate was clarified by using the average intensity of the very faint band that showed up in that lane after the implementation of background subtraction (Table 2, Figure 2). This revealed that the band intensity for the 60kDa Akt band for the tissue lysates showed a lower expression of Akt in the kidney tissue as compared to the expression of the protein in the heart and lung but was slightly higher than the expression of Akt in the diaphragm (Table 2; Figure 2). This was however not conclusive since the band that was seen in the lane of the kidney sample was not clearly defined.
Table 1. Total protein concentrations of lysates of heart, lung, diaphragm and kidney tissues calculated in µg/µl using a standard curve with correlation co-efficient (r) of 0.987. The volumes of the protein extracts loaded for the PAGE were equivalent to 50µg. This was calculated using the total protein concentrations of the various tissue lysates.
TOTAL PROTEIN CONC (µg/µl)
VOLUME (µl) = 50µg
r= 0.987 Abs= 0.738 - concentration + 0.095
Unfortunately, there were other banding patterns on the gel which could have been the result of non-specific binding during the treatment of the blot with the antibodies. The extraneous bands could have also resulted from inadequate blocking and washing during the immunoblotting procedure. Nevertheless, this did not pose a major problem as the band we were considering (Akt 60kDa) was not near the area where the aberrant bands appeared (Figure 1).
SeeBlue® Plus 2
Figure 1. High levels of Akt expression in the heart and lung with relatively low expression level in the diaphragm and no Akt expression in the kidney. To examine Akt expression, equal amounts (i.e. 50µg) of total protein extracted from the heart, lung, diaphragm and kidney of a mouse were subjected to Western blot analysis. The PVDF membrane blot was probed with mouse anti-Akt/PKBα (Cat #BD610861, Becton Dickinson) followed by probing with goat anti-mouse antibody conjugated to IRDye 800® (Cat # 610-132-121, Rockland Immunochemicals Inc). The blot was imaged using the LI-COR® Odyssey Imager at the 800 channel with the incorporation of background subtraction.
Table 2. Band intensities obtained for Akt 60kDa bands in the heart, lung, diaphragm and kidney tissue lysates on Western analysis. The band intensities were determined using the image of the bands on the blot after background subtraction. The image of the gel was obtained using the LI-COR Odyssey® infrared imaging system.
Int INT (k counts)
Raw integ. INT
Figure 2. Relationship between the levels of expression Akt in the heart, lung, diaphragm and kidney of mouse using the average intensities of the bands obtained for 60kDa Akt bands on Western blot image. The image of the blot was taken using the LI-COR® Odyssey Imager via the 800 channel corresponding to the IRDye®800-secondary antibody conjugate.
Akt also known as protein kinase B (PKB) is a serine/threonine protein kinase and has been shown to be targeted by PI3K (2). Akt has also been associated with different cellular processes involving apoptosis (3), cellular regeneration and survival (4), angiogenesis and vascular integrity (5) and diabetes (6). This and other research findings have stimulated even further investigations into the role that Akt may play in the cell and the mechanisms involved in its activities.
We examined the levels of expression of Akt in four different mouse tissues which included the heart, lung, diaphragm and kidney. The results from the Western analysis showed the highest expression of Akt in the heart tissue. The level of expression of Akt in the heart strengthens the fact that Akt signaling may play an important role in the heart either structural or functional. Although our results are not sufficient to attest to this, the cardioprotective role of Akt has earlier on been demonstrated in heart muscle cells and was indicated to improve survival to hypoxia in cardiac muscle cell (7).
In the same vein, the high level of expression of the Akt in the lung (Figure 1) could imply that Akt may play a significant role in the lung. This assumption is in line with the high expression of Akt observed in the lungs of embryonic mice during development (8). This high expression has been associated with the role that the protein plays during lung development, more specifically in the development and preservation of the structure of the alveoli (9). Furthermore, Akt in its activated state protects the lungs from oxidative injury (10). This is very vital to survival especially since any impaired restoration of an injured lung tissue could result in death.
Not much has been reported about the function of Akt in the diaphragm. However, the relatively low level of expression of Akt in the diaphragm (Figure 1) could be linked to the significance of the protein in the diaphragm. For instance, there is the tendency for the diaphragm to atrophy in patients that require mechanical ventilation. This could result in oxidative stress and has adverse effects on the patient. It is interesting to know that in the midst of all these effects there is no change in the regulation of Akt(11).This supports the assumption that Akt might not play a very significant role in the diaphragm.
The lack of expression of Akt in the kidney (Figure 2) was somewhat expected since high levels of endogenous Akt expression have been reported in cases of malignancies associated with the kidney (12). Nonetheless, in the case of a chemical injury to the kidneys as demonstrated in phenol-injected mice, there is no increase or decrease in the expression of Akt in the toxically- injured kidneys (13).
In summary, the results of this experiment were not enough to assert the assumptions that have so far been made. However, Akt expression in the heart, lung, diaphragm and kidney could still be somehow associated with the importance of the protein in these tissues. Also, we were unable to examine the different states (active/phosphorylated or inactive/ dephosphorylated) of Akt in these tissues in order to better understand the function of the protein the tissues. This would be very important in channeling drug therapy to target the protein either to upregulate or to downregulate its expression. This will also enable us to determine how Akt could be targeted in therapeutic procedures in the treatment of cancer or how its positive effects could be harnessed to deal with tissue damage and promote cell survival.
In this experiment, Western analysis was used to determine the expression of Akt in the heart, lung, diaphragm, and kidney. In the future, the expression of Akt in tissues like spleen, thymus and lymph nodes could also be assessed. This will provide a basic understanding of any immunological function that could be associated with Akt. It can be hypothesized that Akt may be involved in certain inflammatory processes that might contribute to cell survival.
To conduct this experiment, the aforementioned tissues will be harvested from a humanely sacrificed mouse. The harvested tissue will be homogenized under cold conditions in liquid nitrogen and then in dry ice to prevent protease activity from breaking down the proteins of interest. The proteins will be extracted from the tissue using ice cold lysis buffer. The homogenate will be separated by the centrifugation and then the supernatant will be saved for polycrylamide gel electrophoresis (PAGE). The total protein concentration of the extracts would be calculated using a standard curve. This will be used to calculate equal amounts of the protein to be loaded onto the polyacrylamide gel. The SeeBlue Plus2 standard (Cat#LC5925, Invitrogen) will be used as a molecular mass standard. The separated proteins after the PAGE will be blotted on to PVDF membrane by electrophoresis. The blot will be blocked with blocking buffer and then probed for Akt using a mouse anti-Akt primary antibody. The blot will then be imaged after treatment with an anti-mouse secondary antibody-fluorescent dye conjugate preferably an IRDye®800 conjugate. At this point, the blot will be shielded from light to preserve the fluorescent dye conjugated to the secondary antibody. Any unbound secondary antibody after this treatment would be washed off with 1X PBS/ 0.1% Tween20. If this is not done, the whole blot would fluorescence under the infrared imager thus obscuring the actual bands of the protein. The blot will be imaged to study the expression of Akt in the spleen, lymph nodes and the thymus.
After running the PAGE there should be well separated ladder-like bands of the molecular mass standard. Also, there could be a single band corresponding to a molecular weight of Akt 60kDa in each of the lanes where the lysates were loaded. Thus, if the western blot depicts a high expression of Akt for any of the lysates it could mean that Akt might be involved in the immunological mechanisms associated with that tissue. This is expected because the localization of Akt in T lymphocytes has been associated with their development and the recognition of antigen presenting cells (APCs) in eliciting immune responses (14). The lymph nodes, spleen and thymus contain an abundance of T lymphocytes among other immune cells. However, the use of the crude extracts from the tissues may not be pure enough to obtain high levels of the proteins of Akt. Also, homogenization may compound the problem by diluting out the protein of interest. Thus, there is the tendency to rule out Akt expression when it is actually expressed in the tissue. Also, because Western analysis is only semi-quantitative, the expression of Akt might not reflect the true quantity of the proteins in the tissues. Furthermore, the expression of Akt might vary with the age, sex or diet of the mouse model to be used for this experiment. Nonetheless, this experiment will give an understanding about how the level of expression of Akt expression is distributed among immune organs like the spleen, thymus and lymph nodes.