Fluorescent Tagged Protein Research Paper

When faced with a crisis of identity, we often stick with those who care in the best position to support us. In Neil Smith’s “Green Fluorescent Protein,” When Max was fighting with Ruby-Doo; he wants to stay with his mom for comfort. GFP is a metaphor for Max’s realization that he is queer. Max is ensuring of his feelings towards Ruby-doo. “In Pop’s arms, the white guinea pig turns a brilliant green…… It’s very weird and really scary. [it’s] kind of beautiful in an unexpected way” [50]. He tried to say same sex love can be weird and scary, but it’s love and love it a beautiful thing. Family plays an important role in max’s life. This is what for example Max’s said “I want to say, don’t go. Not that I’m afraid her date

To make the specimen compatible with both forms of advanced microscopy, they sufficiently prepared samples by coupling the specimen with a fluorescence that was also conductive. This technique was accomplished with the FlouroNanogold label, which contains gold nanoparticles covalently bonded to a fluorescence label. That way, the LM worked as well as the EM for the same set of kinetochores that were being studied. The Hec1 protein was stained in this case because this protein naturally delineates the structures to be studied.

Group 1, the gel picture and Andersons experiment are closely related. The binding did occur, in the 1st lane of group 1the dye was free therefore no binding occurred because no Bovine Serum Albumin (BSA) was present in the mixture. While in lanes 2 to 4 binding did occur because there was BSA in the mixture, with the higest binding occurring in lane 4, this is because there was no buffer to interrupt the binding. (saturation).

Purpose: Green fluorescent protein (gfp) and blue fluorescent protein (bfp) extracted from bacteria were separated by chromatography by means of a molecular sieve matrix. This process utilized the proteins size and shape and was able to separate it from the other molecules and be collected in test tubes. The tubes containing the desired protein will glow under U.V. light. By running an electrophoresis gel with these proteins, one can compare the protein bands and be able to determine the approximate molecular weight of the proteins. In this experiment, students learned about the methods and procedures to partially purify gfp and bfp.

The cells are placed into a flask and are forced through a nozzle so small that they must pass through one by one. In the nozzle, the cells are vibrated at different frequencies to produce drops (3). The drops of cells are then scanned by a laser that is used to count and measure each cell. Separating populations of cells involves attaching antibody linked fluorescent dye to certain cells of interest (3). The information that is gathered from the sorting and measuring of the cells is evaluated by a computer. The final steps for the FACS include applying an electrical charge to the drops of cells (3). Before the drop of cell forms at the end of the nozzle, a charge is applied to the stream that will determine where the drop will go (3). Based on the charge, the drip is either moved left or right with electrodes or placed in to designated final tubes. Quantifying the FACS information involves displaying the information so we know how many cells of each color and charge were

Proteins with his-tags are purified using IMAC, immobilized metal affinity chromatography. The protein product interacts with a metal that is reversibly bound to an immobilized chelating group. The immobolized chelating group acts as a Lewis base (electron-pair donor) to which the Lewis acid (electron-pair acceptor) metal ion is coordinated. The support to which the metal ion binds is called a ligand. When an electron donor group is replaced by another, the action is referred to as ligand exchange. The donor atoms involved in this exchange are the electronegative nitrogen, sulfur, or oxygen. These atoms scavenge for sources of electrons. The structure formed when the metal ions are added to form the chelate result in free coordination

Cameleons, the first generation of GECIs [161], are chimeric proteins that, in their original iteration, were composed of a blue or cyan FP as a FRET donor and a green or yellow FP as a FRET acceptor. The two FPs are connected by CaM and its binding partner M13 (the CaM-binding domain of myosin light chain kinase) (Figure 6A). A similar design with only the CaM-binding domain was also reported in 1997 [162]. When the CaM domain of a cameleon binds to Ca2+, it changes its conformation and sequentially binds to M13, which results in a more compact conformation that decreases the distance between the FRET donor and acceptor. This decreased distance causes an increase in FRET efficiency that manifests itself as

In modern neuroscience, accurate optical reporting of electrical activity in defined neuronal populations can be monumental task. The reason for the enormity of this task is due largely in part to the lack of a sufficient, effective and accurate optical reporting procedure for activity of the brain. At this point in time, genetically encoded fluorescent indicators seem to be the answer for accurate and effective optical reporting, largely because they allow monitoring of defined neuronal circuits and do not require any chemical access. Ideally, these indicators would produce large fluorescent responses, allowing imaging on how input signals are processed into output signals by neurons. In addition, these ideal indicators would have

The primary antibody is the antibody that will bind to the protein of interest. This is generated by exposing the halorhodopsin protein to a rat so that an immune response of antibodies can be generated and these antibodies are harvested from the rat. This antibody will bind to the protein of interest during incubation. The detection, however, is done with the use of a secondary antibody generated from another animal. When unbound primary antibodies are removed from the blotting sheet, the secondary antibody can be used to detect bound primary antibodies. The secondary antibody will be modified with a fluoresce tag that lights up when the secondary antibody binds with the bonded primary antibody. If there is a detectable fluoresce signal, then there is the halorhodopsin protein present in the blot. The amount of fluoresce indicates how much of the halorhodopsin gene is being generated. With the confirmation that HaloX gene was properly inserted into the genome, pain testing with optogenetics can

Living cells should be cultured on petri dishes and then incubated with a tracer in order for downstream flux analysis. Once the metabolic processes have been quenched, the next step is to lyse the cells, separating both the polar and non-polar metabolites from the other cellular substances at the same time. After these steps, the cells are shaken to completely lyse the membranes allowing for a more efficient extraction of all possible biomolecules. After shaking, there should be a clear separation between the polar and non-polar phase for the metabolites, with a well-defined interphase containing proteins and nucleic acids. After centrifugation, the different phases are now separated, and each one can be sampled for further analysis

It is often used in conjunction with epifluorescence and involves the use of long wavelengths to excite fluorophores, which are fluorescent chemical compounds that light up upon excitation, in the tissue of interest (Mempel, 2004). These techniques have shed light on the crawling at a molecular level, including the importance of integrins.

Fluorescence microscopy uses ultraviolet light as its light source that allows the resolution of the object to increase. Fluorescence microscopy contains a barrier filter that reduces any undesired stray of light other than the emission wavelength that might reach the observers eye. Light emitted by the specimen’s fluorophore for viewing is focused and magnified by the objective lens.

It is an automatic whole slide scanner that is comprised of the following components: an optical focusing system, a CCD camera, a Z-focusing system, an XY-translational stage, bright field illumination, a multi-color fluorescence excitation and emission filter system, a microprocessor control board, and a computer. The user can upload up to 8 slides for processing and can view images in up to 5 fluorescent colors. Vanguard scans the entire surface of the microfluidic slide with a series of fluorescence filters that are defined for a given assay and acquires images. A standard assay includes capture of images using filters for DAPI (nucleus), Alexafluor 488 (pan cytokeratin [PanCK], an epithelial marker) and Alexafluor 594 (CD45, a leukocyte marker). Additionally the user can capture data with filters for Cy3, Cy5 or PE and APC. After data acquisition is completed, the images are analyzed for any event where DAPI and cytokeratin are within a specified space in the slide, i.e., indicating the possible presence of a cell with a nucleus that expresses cytokeratin. Images from each fluorescent color, as well as a composite image are presented to the user in a gallery for CTC calls. A cell is classified as a CTC when it is DAPI+, PanCK+ and CD45-. A check mark is placed by the operator next to the composite images and the software tallies all

For cellular biology analyses, I have two years of training in confocal microscopy to perform complex experiments including: Fluorescence Resonance Energy Transfer, Fluorescence Recovery After Photobleaching, real time live cell imaging. These provided the means to analyze both the spatial and temporal properties of interacting proteins using microscopy. Cell

The analysis of a complete set of proteins (proteome) of a given cell or organism can be defined as proteomics (Phillips & Bogyo, 2005). The primary aim of proteomic analysis is to separate, identify and characterize proteins and understand their interactions with other proteins. There are four branches of proteomics which are sequence and structural proteomics, expression proteomics, interaction proteomics and functional proteomics.