Collection of tissue from mouse
C57/BL/6 mice were humanely sacrificed by suffocation followed by exsanguination. Heart, lung, stop and kidney tissues were excised and were instantly transferred to liquid N ( -70 & A ; deg ; C ) and afterwards onto dry ice ( -78.5 & A ; deg ; C ) to forestall peptidases from interrupting down the protein of involvement which in this experiment was Akt. Proteases break down proteins by hydrolysing peptide bonds that link aminic acids in the proteins. The really cold temperatures used in treating the samples in the experiment reduced protease activity and preserved the unity of the Akt, which enabled us to analyze its look in the tissues.
Homogenization of the excised tissues
The tissues were homogenized with the assistance of a tissue homogenizer ( Tissuemizer ) . The full procedure was maintained on ice in order to decelerate down peptidase activity. Besides, complete Minitab ( Cat # 1836153, Roche Diagnostics Corporation, Indianapolis, IN ) was added to the lysis buffer to suppress protease activity. The homogenizer dispersed the tissues into really little pieces in order for the ice cold lysis buffer ( incorporating 1.0 % NP40, 10 % Glycerol, 20mMTrizma, 137mMNaCl, 1mM MgCl2.6H2O, 1mMCaCl2 dihydrate ) to expeditiously lyse the cells to let go of the proteins out of the cells into the lysate. The dust formed deposit after centrifugation at 14000rpm at 4 & A ; deg ; C while the supernatants incorporating the Akt were saved for Western analysis.
Determination of entire protein concentration
Entire protein concentration of the lysates was calculated to find the sum of protein infusion to be loaded on the PAGE gel. By so making, we were able to lade equal sums of the assorted lysates in order to compare their set strengths on the Western smudge. To make this, we employed the usage of the BIO-RAD protein assay reagent ( Cat # 500-0006, BIO-RAD, Hercules, CA ) . This works by the Bradford check rule ( 1 ) which is based on the colour alteration of Coomassie superb blue dye in response to assorted concentrations of proteins. In an acidic medium, the dye binds to basic and aromatic amino acid residues. This causes the ruddy brown dye to alter to a bluish colour whose optical density can be measured with mention to the optical density and concentrations of bovine gamma globulin protein criterions ( Cat # 500-0208, Bio-Rad ) . The optical density was measured at 595nm due to the fact that the difference between the reddish/brown and the bluish colour of the dye is best observed at 595nm. The assorted optical density values for the standard protein samples that were run aboard the samples were used to build a standard curve. The equation of this standard curve was used to cipher the concentration of the protein extracts.
Polyacrylamide gel cataphoresis ( PAGE )
This uses the rule 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 foremost boiled at 90 & A ; deg ; C for 5mins in lading buffer composed of an NP LDS sample buffer ( Cat # NP0007, Invitrogen ) and cut downing agent ( Cat # NP0009, Invitrogen ) . This gel concentration was selected in order to accomplish at least 70 % migration of the protein of involvement through the gel matrix from the point of beginning. The NP sample cut downing agent ( Cat # NP0009, Invitrogen ) contained DTT ( dithiothreitol ) which reduced any disulfide bridges that held together the third construction the proteins in the lysate. The proteins were made to presume an overall negative charge by the handling the lysates with LDS sample buffer ( Cat # NP0007 ) incorporating an anionic detergent LDS ( lithium dodecyl sulphate ) . The LDS edge strongly to and denatured the third construction of the proteins while using an overall negative charge to the proteins in the infusion. Therefore, the proteins in the lysate were made to open up into rod-like shaped constructions with a series of the negatively charged LDS molecules along their polypeptide ironss. Heating the samples to 90 & A ; deg ; C helped to farther interrupt any staying disulfide linkages in the third construction of the proteins. We did this because the LDS and the cut downing agent may non hold wholly cleaved all the disulfide bonds. A hole was poked in the screen of the tubing while the warming was done to forestall the reaction mixture from over boiling. The sample buffer besides contained a trailing dye which helped us to supervise the cataphoretic tally. SeeBlue® Plus2 pre-stained criterion ( Cat # LC5925, Invitrogen ) was run aboard the assorted tissue protein extracts. This criterion was composed of standardization proteins of known molecular weights, including myosin 188kDa, phosphorylase 98kDa, BSA 62kDa, glutamic dehydrogenase 49kDa, intoxicant dehydrogenase 38kDa, carbonaceous anhydrase 28kDa, myoglobin 17kDa, lysozyme 14kDa, aprotinin 6kDa and inulin ? concatenation 3kDa. We compared these sets with that which we obtained for Akt 60kDa to guarantee that the set fit the molecular weight of Akt. Because the criterion was pre-stained we were besides able to cognize the right orientation of the smudge for imaging after reassigning the proteins by electroblotting from the gel on to the membrane.
In this experiment, we used electroblotting to reassign the detached proteins in the gel on to a membrane with high protein adhering capacity such as nitrocellulose or PVDF ( polyvinylidene fluoride ) . An electric current was used to draw the proteins from the gel on to a PVDF membrane. PVDF has a high protein adhering capacity, strong hydrophobicity and dissolver opposition which made it the membrane of pick 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 minimum harm to the protein sets and without any deformation in how they were arranged on the gel. The smudge was labeled with a pencil since bluish ink would shed blood into the smudge and caused intervention during the imaging procedure. This is due to the fact that the LI-COR Odyssey® infrared imaging system detects bluish colour as fluorescence and this interferes with the existent fluorescence produced by the fluorescently- labeled antibodies bound to the smudge.
After reassigning the proteins on to the PVDF membrane we were able to examine the smudge for the protein of involvement Akt utilizing a primary antibody, sneak anti-Akt ( Cat # BD610861, Becton Dickinson, Franklin Lakes, NJ ) antibody which specifically binds to Akt1. Before this, we treated the membrane with Odyssey barricading buffer ( Cat # 927-40100, LI-COR, Lincoln, NE ) to cut down any background intervention and non-specific binding of the primary antibody. The anti-mouse primary antibody was raised in mouse and was designed to acknowledge the Akt1 isoform, irrespective of the province of the Akt, whether it is in its phosphorylated active signifier or in its unphosphorylated inactive signifier. This primary antibody was used to handle the smudge at a dilution of 1:1000 and this was incubated over three yearss since a slow but specific binding is known to give much better consequences. The smudge was washed in 1X PBS ( phosphate buffered saline ) ( Cat # 11666789001, Roche Diagnostics Corporation ) incorporating 0.1 % Tween-20 ( Cat # P5927, Sigma-Aldrich, St Louise, MO ) to take any anti-Akt which did non adhere to the Akt protein on the smudge. The secondary antibody ( Cat # 610-132-121, Rockland Immunochemicals Inc, Gilbertsville, PA ) was raised in caprine animal against mouse antibody. The secondary antibody was conjugated to IRDye®800, an infrared dye which has been optimized for usage on the LI-COR Odyssey® imaging system. During the intervention with the secondary antibody the smudge was protected from visible radiation to continue the fluorescence of the secondary antibody-IRDye®800 conjugate. Therefore, the secondary caprine animal 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 bend edge to Akt on the PVDF membrane. The smudge after intervention with the secondary antibody was washed once more with 1X PBS ( Cat # 11666789001, Roche Diagnostics Corporation ) incorporating 0.1 % Tween ( Cat # P5927, Sigma-Aldrich ) to take any unbound secondary antibody. This helped to avoid the full smudge from fluorescing during imaging. Therefore, merely the countries on the smudge that the secondary organic structure was bound to the primary would demo fluorescence.
Imaging the Immunoblot
The LI-COR Odyssey® infrared imaging system was used to specifically observe the presence of the protein on the membrane by observing the blossoming of an IRDye® 800-secondary antibody conjugate utilizing an infrared optical maser. When seeable visible radiation is used to image, it produces high background and autofluorescence. For this ground, the usage of an infrared optical maser is recommended because it reduces the happening of both autofluorescence and visible radiation spread which produce background. On the Odyssey imager there are two infrared sensing channels that detect fluorescence at 700nm and 800nm with the incorporation of optical filtering. The 100nm difference between these two sensing channels ensures that each sensor measures merely 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 excitement wavelength of 780nm. Therefore, when the infrared optical maser is incident on the IRDye-secondary antibody conjugate on the smudge it caused an excitement in the dye which resulted in a spectral displacement doing the dye to breathe visible radiation at a longer wavelength ( 780nm ) which was detected by the 800 channel infrared sensor.
In this experiment, we examined the look of Akt in the bosom, lung, stop and kidney of a mouse to find if the degree of look of Akt was related to the function that the protein is thought to play in these variety meats. The concentration of entire protein in the tissue lysates from the bosom, lung, stop and kidneys every bit good as the criterions were used to cipher the concentration of the entire protein in each of the tissue lysates. The correlativity coefficient for the standard curve generated was 0.987 which meant that the standard curve could be used to generalize the concentration of the lysates ( Table 1 ) . Therefore, equal sums ( 50µg ) from each of the mouse tissue lysates were loaded on to the acrylamide gel for cataphoresis.
After running the gel and imaging the smudge on the LI-COR Odyssey imaging system, good separated ladder-like sets of the See Blue® Plus 2 pre-stained molecular weight criterion ( Cat # LC5925, Invitrogen ) were observed. In each of the lanes of the lysates from the bosom, lung, and diaphragm tissues there was a seeable individual set which corresponded to the 60kDa molecular weight of endogenous Akt ( Figure 1 ) . The western smudge depicted the look of Akt in the bosom, lung and stop but the look of the protein in the kidney was non evident in the image of the smudge ( Figure 1 ) . The absence of a clear Akt60kDa set in the lane of the kidney lysate was clarified by utilizing the mean strength of the really weak set that showed up in that lane after the execution of background minus ( Table 2, Figure 2 ) . This revealed that the set strength for the 60kDa Akt set for the tissue lysates showed a lower look of Akt in the kidney tissue as compared to the look of the protein in the bosom and lung but was somewhat higher than the look of Akt in the stop ( Table 2 ; Figure 2 ) . This was nevertheless non conclusive since the set that was seen in the lane of the kidney sample was non clearly defined.
Table 1. Entire protein concentrations of lysates of bosom, lung, stop and kidney tissues calculated in µg/µl utilizing a standard curve with correlativity co-efficient ( R ) of 0.987. The volumes of the protein extracts loaded for the PAGE were tantamount to 50µg. This was calculated utilizing the entire protein concentrations of the assorted tissue lysates.
TOTAL PROTEIN CONC ( µg/µl )
VOLUME ( µl ) = 50µg
( 50/30.358 ) -1=1.647? 2µl
( 50/17.529 ) -1=2.852? 3µl
( 50/21.775 ) -1=2.296? 2µl
( 50/88.807 ) -1=0.563? 1µl
r= 0.987 Abs= 0.738 – concentration + 0.095
Unfortunately, there were other banding forms on the gel which could hold been the consequence of non-specific binding during the intervention of the smudge with the antibodies. The immaterial sets could hold besides resulted from unequal blocking and rinsing during the immunoblotting process. However, this did non present a major job as the set we were sing ( Akt 60kDa ) was non near the country where the deviant sets appeared ( Figure 1 ) .
SeeBlue® Plus 2
Figure 1. High degrees of Akt look in the bosom and lung with comparatively low look degree in the stop and no Akt look in the kidney. To analyze Akt look, equal sums ( i.e. 50µg ) of entire protein extracted from the bosom, lung, stop and kidney of a mouse were subjected to Western smudge analysis. The PVDF membrane smudge was probed with mouse anti-Akt/PKB? ( Cat # BD610861, Becton Dickinson ) followed by examining with goat anti-mouse antibody conjugated to IRDye 800® ( Cat # 610-132-121, Rockland Immunochemicals Inc ) . The smudge was imaged utilizing the LI-COR® Odyssey Imager at the 800 channel with the incorporation of background minus.
Table 2. Band strengths obtained for Akt 60kDa sets in the bosom, lung, stop and kidney tissue lysates on Western analysis. The set strengths were determined utilizing the image of the sets on the smudge after background minus. The image of the gel was obtained utilizing the LI-COR Odyssey® infrared imaging system.
Int INT ( k counts )
Raw integ. INT
Figure 2. Relationship between the degrees of look Akt in the bosom, lung, stop and kidney of mouse utilizing the mean strengths of the sets obtained for 60kDa Akt sets on Western smudge image. The image of the smudge was taken utilizing the LI-COR® Odyssey Imager via the 800 channel matching to the IRDye®800-secondary antibody conjugate.
Akt besides known as protein kinase B ( PKB ) is a serine/threonine protein kinase and has been shown to be targeted by PI3K ( 2 ) . Akt has besides been associated with different cellular procedures affecting programmed cell death ( 3 ) , cellular regeneration and endurance ( 4 ) , angiogenesis and vascular unity ( 5 ) and diabetes ( 6 ) . This and other research findings have stimulated even farther probes into the function that Akt may play in the cell and the mechanisms involved in its activities.
We examined the degrees of look of Akt in four different mouse tissues which included the bosom, lung, stop and kidney. The consequences from the Western analysis showed the highest look of Akt in the bosom tissue. The degree of look of Akt in the bosom strengthens the fact that Akt signaling may play an of import function in the bosom either structural or functional. Although our consequences are non sufficient to certify to this, the cardioprotective function of Akt has earlier on been demonstrated in bosom musculus cells and was indicated to better endurance to hypoxia in cardiac musculus cell ( 7 ) .
In the same vena, the high degree of look of the Akt in the lung ( Figure 1 ) could connote that Akt may play a important function in the lung. This premise is in line with the high look of Akt observed in the lungs of embryologic mice during development ( 8 ) . This high look has been associated with the function that the protein plays during lung development, more specifically in the development and saving of the construction of the air sac ( 9 ) . Furthermore, Akt in its activated province protects the lungs from oxidative hurt ( 10 ) . This is really critical to survival particularly since any impaired Restoration of an injured lung tissue could ensue in decease.
Not much has been reported about the map of Akt in the stop. However, the comparatively low degree of look of Akt in the stop ( Figure 1 ) could be linked to the significance of the protein in the stop. For case, there is the inclination for the stop to atrophy in patients that require mechanical airing. This could ensue in oxidative emphasis and has inauspicious effects on the patient. It is interesting to cognize that in the thick of all these effects there is no alteration in the ordinance of Akt ( 11 ) .This supports the premise that Akt might non play a really important function in the stop.
The deficiency of look of Akt in the kidney ( Figure 2 ) was slightly expected since high degrees of endogenous Akt look have been reported in instances of malignances associated with the kidney ( 12 ) . However, in the instance of a chemical hurt to the kidneys as demonstrated in phenol-injected mice, there is no addition or lessening in the look of Akt in the toxically- injured kidneys ( 13 ) .
In drumhead, the consequences of this experiment were non plenty to asseverate the premises that have so far been made. However, Akt look in the bosom, lung, stop and kidney could still be someway associated with the importance of the protein in these tissues. Besides, we were unable to analyze the different provinces ( active/phosphorylated or inactive/ dephosphorylated ) of Akt in these tissues in order to better understand the map of the protein the tissues. This would be really of import in imparting drug therapy to aim the protein either to upregulate or to downregulate its look. This will besides enable us to find how Akt could be targeted in curative processs in the intervention of malignant neoplastic disease or how its positive effects could be harnessed to cover with tissue harm and promote cell endurance.
In this experiment, Western analysis was used to find the look of Akt in the bosom, lung, stop, and kidney. In the hereafter, the look of Akt in tissues like lien, Thymus and lymph nodes could besides be assessed. This will supply a basic apprehension of any immunological map that could be associated with Akt. It can be hypothesized that Akt may be involved in certain inflammatory procedures that might lend to cell endurance.
To carry on this experiment, the aforesaid tissues will be harvested from a humanely sacrificed mouse. The harvested tissue will be homogenized under cold conditions in liquid N and so in dry ice to forestall peptidase activity from interrupting down the proteins of involvement. The proteins will be extracted from the tissue utilizing ice cold lysis buffer. The homogenate will be separated by the centrifugation and so the supernatant will be saved for polycrylamide gel cataphoresis ( PAGE ) . The entire protein concentration of the infusions would be calculated utilizing a standard curve. This will be used to cipher equal sums of the protein to be loaded onto the polyacrylamide gel. The SeeBlue Plus2 criterion ( Cat # LC5925, Invitrogen ) will be used as a molecular mass criterion. The detached proteins after the PAGE will be blotted on to PVDF membrane by cataphoresis. The smudge will be blocked with barricading buffer and so probed for Akt utilizing a mouse anti-Akt primary antibody. The smudge will so be imaged after intervention with an anti-mouse secondary antibody-fluorescent dye conjugate sooner an IRDye®800 conjugate. At this point, the smudge will be shielded from visible radiation to continue the fluorescent dye conjugated to the secondary antibody. Any unbound secondary antibody after this intervention would be washed off with 1X PBS/ 0.1 % Tween20. If this is non done, the whole smudge would fluorescence under the infrared imager therefore befoging the existent sets of the protein. The smudge will be imaged to analyze the look of Akt in the lien, lymph nodes and the Thymus.
After running the Page there should be good separated ladder-like sets of the molecular mass criterion. Besides, there could be a individual set matching to a molecular weight of Akt 60kDa in each of the lanes where the lysates were loaded. Therefore, if the western smudge depicts a high look of Akt for any of the lysates it could intend that Akt might be involved in the immunological mechanisms associated with that tissue. This is expected because the localisation of Akt in T lymphocytes has been associated with their development and the acknowledgment of antigen showing cells ( APCs ) in arousing immune responses ( 14 ) . The lymph nodes, lien and Thymus contain an copiousness of T lymph cells among other immune cells. However, the usage of the petroleum extracts from the tissues may non be pure plenty to obtain high degrees of the proteins of Akt. Besides, homogenisation may intensify the job by thining out the protein of involvement. Therefore, there is the inclination to govern out Akt look when it is really expressed in the tissue. Besides, because Western analysis is merely semi-quantitative, the look of Akt might non reflect the true measure of the proteins in the tissues. Furthermore, the look of Akt might change with the age, sex or diet of the mouse theoretical account to be used for this experiment. However, this experiment will give an understanding about how the degree of look of Akt look is distributed among immune variety meats like the lien, Thymus and lymph nodes.