Expression Profiling of a Novel Protein
A new transcription unit was discovered while working with transfected murine BAC clones, because a novel spot appeared on a 2-dimensional protein gel. Through a process of expression subcloning from the BAC clone, the transcription unit that generated the novel peptide was located. This finding was back validated by sequencing the protein contained in the 2-D gel piece using N-terminal Edman degradation and mass spectroscopy (Rosenfeld, Capdevielle, Guillemot, and Ferrara, 1992; Hellman, Wernstedt, Gonez, and Heldin, 1995).
This transcription unit happens to be encoded within a multi-gene locus that is coordinately regulated in a tissue-specific and developmental manner. Since the laboratory is already heavily invested in characterizing the gene regulatory mechanisms that control this locus, and the downstream roles of the gene products, understanding the expression pattern of this novel gene may be important to ongoing research efforts.
Determination of mRNA patterns of expression
In addition to gearing up for producing this protein for both polyclonal and monoclonal antibody production, a substantial amount of information can be obtained by analyzing the tissue expression pattern(s) of the RNA transcript that encodes this protein. Commercial RNA preparations are available that allow rapid screening of mouse tissues (for example see ClonTech, 2011), including embryonic stages, using RT-PCR. If the gene is suspected of being expressed at low levels then a real-time PCR approach will be utilized. Additional controls will be needed, including the quantification of three to five housekeeping gene targets to control for any variations in reverse transcriptase reaction efficiency between tissue samples (Xu, Ma, Cui, Li, Ning, and Wang, 2010). In addition, yeast RNA will be used to control for any sample contamination issues that could arise (negative control).
Once the tissues and developmental stages expressing the transcript has been established using RT-PCR, then the location within mouse embryos and tissues can be further refined using in situ hybridization (Yan et al., 2010; Carter, Fletcher, and Thompson, 2010). Tissues suspected to be expressing the transcript are fixed in paraformaldehyde and embedded in paraffin wax. Microtome slices are prepared and allowed to dry on glass slides. After removing the wax, the tissue slices are blocked with a solution containing yeast RNA and Denhardt's reagent (milk protein, carbohydrates, and vinyl polymers), and then incubated with the fluorescently-tagged RNA probe. After several washings, RNAse A treatment, and H&E counter-staining, the location of the probe within the tissue is visualized and captured digitally using fluorescent and light microscopy. Should the fluorescent probe fail to provide a good signal-to-noise ratio, radioactive RNA probes will be used.
Determination of protein patterns of expression
The development of an antibody probe can be subcontracted out for a sizeable fee, or bacterial-expressed peptides can be injected into rabbits and mice in-house in an effort to generate polyclonal (Cooper, and Paterson, 2008) or monoclonal (Yokoyama, 2006) antibodies, respectively. Confirmation of the specificity of the antibody can be accomplished by probing Western blots containing protein extracts from known positive and negative controls (Gallagher, Winston, Fuller, and Hurrell, 2008). A positive control can be created by stably transfecting an expression construct into the mouse fibroblast 3T3 cell line together with a selectable marker like the neomycin resistance gene, expanding colonies in G418-containing medium, and preparing protein extracts from the expanded clones (Mortensen and Kingston, 2009). Bacterial protein extracts can serve as a negative control, unless prior RT-PCR screening of available murine cell lines revealed one or more are non-expressing. Should background problems occur the antibody will be affinity-purified. Should any of the antibodies prove to work well on Western blots, protein extracts from tissues expressing and not expressing the transcript will be screened by Western blot.
The figure to the right shows a dilution series determining the optimal antibody concentration for low background. Lanes are 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, 1/6400 from left to right. The molecular weight ladder on the right goes from 200, 116, 97, 66, 43, 24, and 18 kDa (Gallagher, Winston, Fuller, and Hurrell, 2008).
If the antibody performs well in the Western blotting of murine tissues, then it will be used to probe tissue sections for the presence of the novel protein using fluorescent microscopy. Tissue sections are blocked with milk protein, washed, incubated with the antibody in blocking solution, and then washed repeatedly. The tissue sections are then incubated with a secondary antibody conjugated to a fluorescent chromophore or horseradish peroxidase. Sections will be counterstained with H&E or other appropriate stains or primary antibodies. Visualization is by fluorescent or light microscopy, respectively, after incubating with fluorescent chromogen-tagged secondary antibodies (Daneshtalab, Dore, and Smeda, 2010).
Should the antibody perform well in Western blots and as an immunohistochemistry probe, then the subcellular location of the novel protein by confocal laser...
G Protein-Linked Receptors An organism must respond appropriately to its internal and external environments day after day in order to survive. The organism's cells respond to internal and external stimuli much like tiny computers that process numerous inputs and also produce numerous outputs in daily existence (Kennedy 2004). These stimuli are signals that come from the general environment or the cells of other or co-existing organisms, proximate or distant, and this
Tumor Suppression Protein 53 and Effects on Cellular Function and Aging Tumor Suppression Protein 53 (P53) was originally linked to tumor proliferation and thought to be an oncogene. Research led to an understanding that P53 was in actuality a tumor suppression protein and suppression of the P52 gene is the most widely genetic defect found in tumors (Levine 1991). Further study of p53 and the family of tumor suppression genes led
In the tissue culture, they usually proliferate indefinitely. The normal constraints which limit the growth of the cells absent in the cancerous state and are also characterized by the division ability for number of generations which is unlimited. Cell cycle and cancer With millions of chemical reactions taking place concurrently and in specific areas, the human body can be thought of as a small laboratory. It is the only "machine" with
The failure of human hamster fusion in the presence of anti-human izumo antibody clearly showed that Human izumo protein is essential for fertilization. This study however has raised some new questions. The difference in fertilization capacities between wild type and Izumo +/- type was not clearly discussed. Also, the fact that ICSI of izumo -/- produces twice as many litters as did the izumo +/- type implicates the possible role
These proteins include homologous members of yeast. The presences of these proteins suggest that E. histolytica is skilled to perform homologous recombination, which is the same as in other organisms. DNA damage was evaluated by TUNEL assay. In yeast and in human cells, histone H2AX becomes rapidly phosphorylated when DSBs are introduced into chromatin (Lavi et al.). Studies show that histone as a protein plays a significant role in the
Nutrigenomics is an important field of study. It finds in roots in modern times, because of the direct relation to advances in science and technology. Nutrigenomics also straddles the nature vs. nurture divide. The publication of the relatively preliminary results of the Human Genome has given greater impetus to the idea of Nutrigenomics. One might assuredly say that the publication of the Human Genome is preliminary because the current versions
Our semester plans gives you unlimited, unrestricted access to our entire library of resources —writing tools, guides, example essays, tutorials, class notes, and more.
Get Started Now