The purpose of this experiment is to illustrate how different immuno-techniques can be carried out and the rationale behind it. All immuno-techniques in this experiment involved the use of antibody to detect the protein of interest. In western blot, a secondary antibody attached with horseradish peroxidase enzyme was used to show the location of the bands indicating molecular weight marker and antigen. For ELISA, a conjugated antibody was used in order to enable the samples to show different levels of absorbance. Via the usage of all these immuno-techniques, the antigen’s molecular weight, concentration of antigens, types of cells presented and their relative amount in the brain as well as the amount of the T-cell of different maturation state presented in different organs were determined. All these proved that immune-techniques were capable of determining the various aspects of the protein of interest and hence, highly useful for the studying of molecular cell biology.
This report mainly focuses on the utilizing of immuno-techniques to detect and analyze various aspects of the proteins of interest presented in the cells. All protein presents in the body play important roles in the proliferation, growth, survival as well as death of the cells and any abnormalities in them can contribute to illness or even death. In order to understand the various aspects of proteins, several immune-techniques have been invented and most, if not all, make use of the specificity of antibody towards an antigen to detect the presence and the amount of a particular protein of interest in samples. Depending on the immuno-techniques employed, the primary antibody can be labeled with a marker molecule which can be an enzyme or fluorescent dye in order to visualize the antibody-antigen complex. A secondary antibody which can bind to the primary antibody can also be labeled with a marker molecule for the same purpose. In this experiment, a total of four types of immune-techniques were carried out in order to understand the processes involved as well as the rationale behind them.
2. Experimental procedure:
2.1 Western Blot
At the beginning of this experiment, primary antibody (anti β-tubulin) solution was added onto the membrane and the lid was put on the Petri plate. The Petri plate was then returned to the rocking platform and was incubated for 45 minutes. This was followed by transferring of the primary antibody back into its container. Next, the membrane was washed with phosphate buffered saline containing Tween20 (PBST) for 2 minutes and this was repeated two more times. After this, secondary antibody solution was added to the membrane and the membrane was rocked for 30 minutes on the rocking platform. The membrane was then washed with PBST for a total of 3 times with each wash lasting for 2 minutes. After washing, the membrane was washed again with PBS for 5 minutes. Next, TMB/B (2~3ml) was poured over the membrane and the membrane was then agitated over a white background for approximately 10 minutes. The TMB/M was then tipped out and the membrane was washed with distilled water 2 times. The membrane was then air-dried and labelled, followed by determination of protein size. Finally, a photo session of the membrane was carried out at the end of the experiment.
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Mouse LIF standards, control and samples obtained from mouse brain homogenates as well as a mouse LIF polyclonal antibody pre-coated plate were prepared before the experiment. First, 50μL of Assay Diluent was added to each well, followed by the addition of 50μL of standard, control or sample to each well. After this, the plate was tapped gently for 1 minute and sealed with adherence tape before being incubated at room temperature for 2 hours. After the 2-hour incubation, each well was washed 5 times with 400μL of wash buffer. Next, the plate is inverted and propped against a clean paper towel. 100μL of diluted mouse LIF conjugate was then added into each well and the plate was incubated at room temperature for another two hours. Following the incubation, each well was then rinsed 5 times with 40μL of wash buffer. After the wash, the plate was inverted and propped against a clean paper towel. This was followed by the addition of 100μL of substrate solution (TMB) into each well. The plate was then gently tapped and sealed with adhesive tape. After this, the plate was incubated at room temperature for 30 minutes. 100μL of stop solution was added into each well following the incubation and the plate was gently tapped. Finally, the optical density of each well was determined within 30 minutes by using a plate reading spectrophotometer with the measurement wavelength set at 450nm and the wavelength correction at 540nm.
The molecular weight of the band of the antigen was estimated to be slightly above 50kDa via observation of the membrane photo [see Appendix 1]. The molecular weight of the antigen was determined to be 53.7kD from the interpolation of the standard curve constructed using the standards [see Appendix 2].
From the standard curve contructed [see Appendix 3], the concentration of the mouse LIF positive control sample was determined to be 200pg/ml while the unknown sample’s concentration was determined to be 140pg/ml. Besides this, there was colour change from green to yellow for all the solution after the stop solution was added and all solution in the wells had varying colour intensity.
3.3 Inspecting immuno-micrographs
3.3.1 Dentate gyrus of mouse
The micrograph A [see Appendix 4] which showed the dentate gyrus of mouse stained using an antibody recognizing all nerve cells showed a large number of yellow dots (neuronal cells) which were mostly clustered together, forming a horn-like shape. On the other hand, in the micrograph B which showed the dentate gyrus of mouse stained using an antibody recognizing only neural stem cells, only quite a small number of scattered red dots (neural stem cells) were observed. However, most of the neural stems cells appeared to exist within the horn-like cluster of neuronal cells as in micrograph A.
3.3.2 Embryo brain
In micrographs A-C [see Appendix 5] which showed the subventricular zone of CREB+/+ brains (wild-type embryo brains), there appeared to be significantly less lateral ventricle space (black space) in-between the cells compared with micrographs D-F [see Appendix 5] which showed the CREB-/- brains (CREB mutant brains). Besides that, the nestin expressing cells (green) in micrographs A-C also appeared to have longer dendrite-like projections as well as more widespread branching than those in micrograph D-F. The other differences between micrographs A and D were that the amount of nestin expressing cells seemed to be larger and having longer projections in micrograph A than in micrograph D. Moreover, the number of cell bodies (blue) in micrograph A was more than those in micrograph D. For micrographs B and E, the other differences between them were that the amount of nestin expressing cells and cell bodies were both larger in the former compared with the latter. On the other hand, the amount of the β-tubulin protein (red) in both micrograph C and F appeared to be almost the same, with the cell bodies and nestin expressing cells presented in larger number in micrograph C than in micrograph F.
3.4 Analysing FACS results
4.1 Western Blot
In this experiment, the primary antibody (anti β-tubulin) solution was added to the membrane at the very beginning in order to form antibody-protein complexes to which secondary antibody attached with the horseradish peroxidise enzyme can bind and convert the colourless substrate into blue coloured product. Besides this, the membrane was subjected to washing several times with PBST, PBS and distilled water to obtain clean blot which enabled clear view of the bands presented on the membrane. PBST helped in washing off the unbound primary antibody from the membrane without washing off the antigen and decreasing the binding of the antibody to unspecific proteins which in return, reduced the background signal of the membrane (1). PBS was later used instead of PBST as the Tween20 in PBST interfered with the horseradish peroxidase reaction which was responsible for converting the colourless substrate into blue-coloured product (2). In addition, each wash lasted for several minutes because the interactions between the bindings of the protein (antigen) to the antibody were occurring throughout the membrane’s thickness, making thorough soaking and washing necessary. This ensured that all unbound antibody was washed off after each round of washing.
There were two missing molecular marker indicators in the membrane [see Appendix 1] and the reason behind this might be that there were insufficient amount of the proteins with the molecular weight of 10kDa and 15 kDa within the molecular marker indicators, resulting in the two bands that represented those two proteins to be too light to show clearly on the membrane.
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Due to the fact that proteins with higher molecular weight moved slower down the gel compared with those with lower molecular weight during the gel electrophoresis, the estimation of the molecular weight of the antigen can be carried out. Besides that, the actual molecular weight of the antigen can be determined via the interpolation of the standard curve [see Appendix 2] because the distance travelled by the proteins is inversely proportional to their molecular weight.
The microtitre plate’s wells were coated with specific antibody which was immobilized and incubation was done after the addition of the samples to ensure that the antibody had sufficient time to bind to the antigen. Washing of the plate was carried out so as to wash off the unbound antigens presented in the samples to increase accuracy of the sample absorbance measurement at the end of experiment. Mouse LIF conjugate was then added to bind with the antibody-antigen complex formed in such a way that the antigen was sandwiched between the antibodies, to convert the substrate (TMB) into blue coloured product to enable detection of the optical density of each well by the plate reading spectrophotometer. Stop solution (diluted hydrochloric acid) was added into the samples in order to stop any further reaction between the TMB and the mouse LIF conjugate so that any further colour change of the samples during the determination of their optical density was prevented. The blue-coloured product from the enzymatic reaction was turned into yellow colour by the stop solution. As the absorbance of the samples and the concentration of mouse LIF antigen presented in the samples are directly proportional to each other, the mouse LIF antigen concentration in the unknown sample can be determined.
4.3 Inspecting immuno-micrographs
In order to obtain immune-micrographs, there were some necessary processes which were required to be carried out. First of all, primary antibody for the antigen of interest was applied to the slides containing the sections of dentate gyrus of mouse and embryo brains. This was to allow the protein of interest to be bound to by the primary antibody which in return, will be served as a target for the gold-labelled/fluorescence secondary antibody to bind to. After about half an hour of incubation, gold-labelled/fluorescence-labelled secondary antibody directed to the primary antibody were then added to the slides so that the proteins of interest can be visualised as the complexes formed exhibited reddish signals (3) and observed in light microscopy or fluorescence microscopy [see Appendix 4].
There are antibodies that are able to detect specific types of cell or to standard cellular structures (4). These antibodies can be used with the other antibodies, especially those which were of different species origins in the same slide. For those antibodies raised in the same species, they must be of different igG isotypes and if not, haptenylation of the antibodies must be carried out so as to avoid cross-reactions between each of the other primary antibodies (4).
4.3.1 Dentate gyrus of mouse
The type of antibodies that was used in micrograph A was polyclonal antibody while the one that was used in micrograph B was monoclonal antibody and they were detected via the use of immunogold technique (3). For micrograph A, the polyclonal antibody was able to bind to all the antigen present on the neuronal cells and by adding the secondary antibody with gold probes attached, all the neuronal cells was stained and hence, visible under light microscope. For micrograph B, the monoclonal antibody was only able to bind to the antigen presented only on the neural stem cells and hence, only neural stem cells were stained when gold-probed secondary antibody was added.
4.3.2 Embryo brains
The nestin expressing cells in CREB-/- brains expressed shorter projections as well as less complex branching were because of the lack of CREB which regulates many important genes responsible for the survival of cell and its function (5). Moreover, the survival rate of the cells which were lacking CREB was greatly reduced at 24 hours, contributing to the reduced neurosphere formation 5 days later (5). Therefore, the amount of cell bodies as well as nestin expressing cells in CREB-/- brains was lesser than those in CREB+/+ brains.
4.4 Analysing FACS results
From the tabulated FACS results as shown in table 3, majority of the T-cells in the thymus were the intermediate matured ones and majority of the T-cells in the spleen as well as lymph node were the least matured ones. This applied to both control and the mutant samples, showing that the development of the T-cells was not affected by the lacking of CREB transcription factor (6).
The result shows that majority of the T-cells in thymus were intermediately matured. The reason behind this was that the intermediate matured T-cells’ production rate exceed the rate of production and exportation of fully matured cells out of thymus, contributing to the higher amount of intermediately matured T-cells in thymus (7). The majority of the T-cells presented in spleen were the least matured T-cells because there were T-cell precursors (least matured T-cells) that were originated from the spleen itself, not from thymus. In the case of lymph node, there were more fully matured T-cells due to the migration of those matured T-cells from the thymus to there.
Various aspects of the proteins of interest can be determined through the usage of the 4 immuno-techniques in this experiment. By understanding more about the proteins, new discovery about how certain diseases work can be made and more effective ways of treatment can be made as well.
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