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The western blot is one of the most utilized techniques in molecular biology for detecting a protein among various tissue, such as in plant cell extracts. The process is relatively simple and cheap to run, and has provided supporting evidence for the role of specific proteins during plant reproduction mechanisms. The name western blot, also referred to as an immunoblot, is a derivative of the southern blot which was termed by E. W. Southern in 1975. The basic procedures of a western blot involve separating native or denatured proteins according to isoelectric point, electric charge, or molecular weight via gel electrophoresis. The separated proteins contained in the gel are transferred to a membrane composed of either polyvinyidene fluoride or nitrocellulose because of their non-specific amino acid binding affinities. The gel and membrane are "stacked" between two filter papers, and the proteins transfer by either application of an electrical field (electroblotting) or capillary action. The membrane is probed most commonly in two steps, resulting in labeled target proteins for detection. The membrane is incubated with primary antibodies specific to the target protein, and any unbound antibodies are removed with washing buffer. A second incubation period involves the binding of secondary antibodies to the target primary antibodies, followed by another washing step. Secondary antibodies are typically linked to a reporter enzyme such as alkaline phosphatase or a peroxidase, which catalyze a reaction to produce a visible dye. Colorimetric detection, a popular visualization method, utilizes a reporter enzyme bound to secondary antibodies as previously mentioned. The reporter enzyme reacts with a soluble substrate during the second incubation period, producing an insoluble, colored precipitate beside the enzyme. The insoluble dye stains the membrane on all locations containing the reporter enzyme and therefore the target protein. Chemiluminescent detection is another popular method that requires a reporter enzyme bound to secondary antibodies, and produces a luminescent signal when the substrate reacts with the enzyme, captured by CCD cameras or photographic film. Other visualization techniques such as radioactive and fluorescent detection do not require exposure to secondary antibodies, and instead contain labeled primary antibodies.
The tagging of a specific antibody to the protein can only be accomplished after the lysis of the cell, making sure the target itself is not denatured. The components of the cell, such as organelles and proteins, are sorted by density, usually through the use of a centrifuge. This allows the larger material to be removed for greater accuracy and reduced "noise". The mixture of proteins is separated based upon molecular weight by running a gel electrophoresis. An electrical current is sent through the gel, which is commonly made from a polyacrylamide, and has a charge that reacts with the proteins to bind and travel across the gel based upon their molecular charges. Protein structure and charge determines the distance travelled, and is very useful for comparison between various genotypes. A molecular ladder can be run alongside each lane to give a reference point for the size of each protein being studied.
The proteins are then transferred to a membrane using a "sandwich" capillary system of certain layers. The gel and membrane are placed between filter paper, and a buffer causes the proteins to travel onto the membrane. Stacking the layers in the fashion is similar to making a sandwich, hence the name. One problem that can arise occurs when antibodies bind to non-specific proteins in the membrane, termed as a false-positive. This is prevented by adding a small amount of unrelated protein, typically Bovine serum albumin (BSA), which "fills in" the areas on the membrane that do not have any bound protein of interest. Once the specific antibodies are added, the only locations it can bind are to the antigens of target proteins. This blocking step is crucial for maintaining accurate results. The use of antibodies during the detection phase of a western blot occurs through a 2 phase process, known as "probing". Primary antibodies are added to the membrane in the first step and incubated for a period of time. Unbound antibodies are washed away, and secondary antibodies or "conjugates" are added to the membrane, which can bind multiple copies to a single primary antibody. A coupled enzyme carries out the reaction which ultimately produces chemiluminescence of bound proteins, and displays the results that researchers are looking to obtain. Multiple processes allow the visualization of the target molecules such as colorimetric, radioactive, and fluorescent detection. Labeled probes are visualized through these techniques upon the addition of a substrate, which reacts with the covalently bound enzyme in the secondary antibody, providing an antibody-mediated image.
In the critical paper created by Stein et al. (1996), a western blot demonstrates the specificity of the S locus by analysis of SRK6 and SLG6 protein. SRK6 is hypothesized to play a role in the SI system displayed by Brassica oleracea, so researchers used SDS-PAGE membrane to compare the SLG6 like domain of the SRK6 protein. A MAb/H8 monoclonal antibody, previously known to recognize this domain, was used to probe the membrane of a GST (glutathione S-transferase) control versus a GST fusion with SRK6. Results displayed that MAb/H8 did recognize the similar domain of 66kD through the use of chromogenic substrates. Proceeding this study, authors used this monoclonal antibody again for another western blot to verify the specificity of the S locus, in that the S6 allele is quite dissimilar to the S2 allele. The MAb/H8 did not recognize the SRK2 fusion protein while again recognizing the SRK6 fusion. The immunoblot provided useful information to this research paper in confirming the specificity of the S locus and correlating proteins.
There are few limitations when using this technique in molecular biology, which demonstrates the overall positive utilization in studying plant reproduction. One of the limitations of this technique is due to the incubation period after the addition of both antibodies. Primary antibodies added to the membrane require a timeframe of 30 minutes up to overnight incubation, and the second period varies among a few hours. Immunoblots can be a long process if overnight incubation is necessary, which can drag out experiments. Another limitation arises from the need for specific antibodies. Mass corporations produce antibodies for sale, however they can be quite expensive. Not all antibodies are mass produced as well, which means one would need to be created or a substitute protein specific to a known antibody must be used. These issues can create slight problems, but the technique is beneficial in the long run.
Immunocytocehmistry is the technique that allows researchers to detect if a protein or peptide of choice displays a particular antigen. This is a crucial process for many biological studies, such as in the previously mentioned western blot. The immune system functions by targeting foreign epitopes to be removed, cleansing the organism with various immune cells. Researchers take advantage of this function precisely through the use of detectable antibodies that will bind to the epitopes of the target peptide or protein. Once the target is tested positive for the presence of the antigen, the sub-cellular localization can be identified through the tag placed on the antibody. Detection of antibodies can be performed in various ways, both indirectly and directly. As in the western blot, the signal becomes amplified upon the binding of secondary antibodies or "anti-serums". A covalently attached enzyme, usually alkaline phosphatase, cleaves a substrate that produces a colored product when added to the cell. A more direct approach utilizes a visible tag fused to a primary antibody. The tag, commonly a gold particle or fluoresced molecule, can be visualized directly through a microscope so there is no need to use secondary antibodies for amplification.
In the research paper published by Escobar-Restrepo et al. (2007), the FERONIA protein indicates the female role of chemotactic signaling during plant reproduction. The FER gene contains a nucleotide sequence responsible for directing the male pollen tube to the female gametophyte, and functions as a receptor-like kinase that is located asymmetrical to the synergid cells. Immunocytology displays this mechanism by comparing antisense probes to sense probes of reproductive cells in mutant and wild type Arabidopsis thaliana. A cDNA probe is labeled with digoxigenin, which provides the binding site for the antibody. The probe is a complementary strand of DNA specific to the mRNA of the FER gene. The antibody binds to the digoxigenin-conjugated dTTP, conjugated with alkaline phosphatase. The conjugated enzyme cleaves a phosphate from the colorless substrate to produce a dark purple dye. FER mRNA is visualized using standard microscopy in all antisense probe lanes that contain female reproductive tissue, along with immature pollen grains. No mRNA is detected in mature pollen, agreeing with the hypothesis as to the role of female specificity in directing the pollen tube to the female gametophyte. Immunocytochemistry also reveals the asymmetrical localization of FER to the synergid cells upon analysis of the FER promoter. The promoter was fused to a bacterial uidA gene and developed using a chromogenic substrate. The results show a concentration of the FER promoter within synergids near the filiform apparatus.
Over the past few decades, fluorescence microscopy has grown to become the primary technique in visualization of cells and their components. Radioactivity had been most commonly used, however it has many limitations and hazards. Fluorescence is a type of luminescence, meaning that the visible light produced occurs without the radiation of energy; no heat is given to the environment. A fluorophore is a fluorescent molecule that emits light, which is both naturally occurring in certain organisms and commercially produced for use in molecular and cell biology studies. The functional group captures energy as an electron travels from a low energy state to an excited state, and releases the energy as the electron moves back down. Energy is absorbed and released as specific wavelengths, which allows the visualization of emitted light.
Among all of the fluorophores in circulation today, green fluorescent protein (GFP) is responsible for revolutionizing fluorescence microscopy. Discovered naturally in the jellyfish species Aequorea victoria, GFP displays a natural autofluorescence which researchers took complete advantage of. The GFP gene can be isolated and combined with the gene of a target cell component, while still allowing normal cell functions to take place. This combination is termed a GFP fusion protein, due to the fused state of each gene. Upon excitation, the GFP chromophores emit the green fluorescent color in all areas of the cell that contain the fusion protein. Blue light is needed to excite GFP proteins because the wavelength emitted is longer than the absorbed energy, and blue has a shorter wavelength in the visible spectrum. Researchers use this protein primarily for sub-cellular localization of target cell components. Since the discovery of GFP, many similar derivatives and fluorescent dyes have been synthesized for use in biological research, with colors ranging the entire visible spectrum. Visualization of fluorescence is obtained by the use of a fluorescent microscope. Specific filters block out unwanted "noise" while a CCD camera or other photosensors capture the luminescence.
Subcellular localization of the FER protein is detected with a GFP-FER construct, displaying localization to both the plasma membrane and filiform apparatus (Escobar-Restrepo et al., 2007). FER is thought to function as a receptor like kinase (RLK) which indicates that it is a transmembrane protein, with both intracellular and extracellular domains. The receptor will activate the phosphorylation of intracellular molecules; possibly leading to a phosphorylation cascade that aids the pollen tube to the female gametophyte. The GFP fusion protein contains the FER promoter sequence, which is tested against a 35S promoter fusion protein known to display non specific localization. Onion epidermal cells were exposed to each construct and viewed under confocal laser scanning microscopy and epifluorescent microscopy, confirming the localization to the plasma membrane with the FER promoter construct, and whole-cell fluorescence of the 35S promoter. Identical results are also shown through fluorescent microscopy of Arabidopsis thaliana leaf epidermis exposed to each construct. These results are consistent with the indication of FER functioning as a RLK protein. An unfertilized ovule and the micropyle area from the same species was analyzed with the same construct. A concentrated area of fluorescence is detected in the filiform apparatus of the synergid, along with noticeable GFP signals on the synergid membrane. Sub-cellular localization to these areas of the synergid is consistent to FER playing a role in pollen tube and female gametophyte reception. The use of fluorescence microscopy supplied valuable data for the female role, specifically FER protein, of signaling pollen tube reception.