The article “Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein” was written by Shiyong Wu and published in the World Journal of Biological Chemistry. The article was published on October 26, 2010, and appears in volume 1 and issue number ten of the journal. The article consists of seventeen pages total and can be found on pages 313 through 320.
The aim of the research was “to study the localization and function of a eukaryotic initiation factor 2 (eIF2)-associated 67-kDa glycoprotein (p67)” (Wu, 2010, p. 313).
Several steps were taken in preparation to localize the samples through the Western blotting method. First, the COS-1 cells were cultured in a medium that contained 10 percent fetal calf serum and penicillin (2010, p. 314). The MCF-10A cells were cultured in a serum-free medium that consisted of insulin, hydrocortisone and epidermal growth factor (2010, p. 314). The SUM102 cells were cultured in a serum-free medium that consisted of insulin, hydrocortisone, and EGF (2010, p. 314). Finally, the SUM149 cells were cultured in a serum-free medium that consisted of insulin and hydrocortisone (2010, p.314). This enabled the researchers to obtain cell samples in a controlled environment for the experiment.
To conduct the Western blotting analysis, researchers resolved the cell extract and proteins by SDS-PAGE and then electroblotted them onto nitrocellulose membranes (2010, p. 315). The nitrocellulose membranes were treated with Tris-buffered saline, which contained 2 percent non-fat dry milk (2010, p. 315). Additionally, the membranes were blotted with an anti-p67 monoclonal antibody before being developed using a solubilized alkaline phosphatase substrate (2010, p. 315). These steps enabled researchers to localize the proteins under examination and observe their functions.
As Figure 4A illustrated, the Western blotting analysis determined two bands in the 67 kDa region (2010, p. 317). The figure shows the appearance of the sample after the total cell extracts, supernatants, and S100 pellets were resolved by SDS-PAGE (2010, p. 317). The blotting analysis revealed that glycosylation was required in order for p67 to bind with ribosome-dense parts of the cell (2010, p. 317). This finding enabled the researcher to elaborate on the behavior of p67 during protein kinase.
This research is significant because the misregulation of protein synthesis kinases is thought to contribute to cancer, thereby opening up a possibility for cancer prevention strategies (2010, p. 314). Previous research indicated that p67 plays a protective role for cells and can aid in regulating cell transformation (2010, p. 314). However, while less research was conducted that attempted to localize the protein and observe its function in protein regulation (2010, p. 314). Thus, the results from this research fill the gap by using several methods to localize p67 so that their function can be determined.
Serving as controls the researchers localized the sample proteins from living organisms through P-labeling in conjunction with immunoprecipitation and Western blotting (2010, p. 313). Further, this enabled them to measure PKR autophosphorylation, eIF2 phosphorylation, and p67 expression in order to make a comparison between normal and breast cancer cells. This enabled the researchers to compare the results in cancerous and noncancerous cells.
Wu, S. (2010). Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein. World Journal of Biochemistry, 1(10), 313-320. doi:10.4331/wjbc.v1.i10.313