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Electrophoretic mobility shift assay - Lab Report Example

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Electrophoretic Mobility Shift Assay Name of Authors Abstract: Determination of DNA-binding proteins is done through the method of Electrophoresis Mobility Shift Assay (EMSA). Specificity Protein 1 (SP1) and other sp members come from family of transaction…
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Download file to see previous pages Results showed that sp1 demonstrated the expression in cancer cells. 1-Introduction The Electrophoresis Mobility Shift Assay gives the simple and rapid method of detection of DNA-binding proteins. Study of transcription factor is another important feature of this method of Electrophoresis Mobility Shift Assay. Use of gel shift assay is more common for complex mixture of proteins or for the incubated and purified proteins. Protein interaction process was studied two decades ago that provided an efficient, and most valuable method to study the protein interactions. It became a powerful method due to its versatility and easier use for the high sensitivity. This method was also useful for the studying the replication, recombination and repairing of DNA. The EMSA added value for kinetic and quantitative analysis. Because of high sensitivity of this method, it became possible to settle the complex issues of DNA or protein stoichiometry. Specificity Protein 1, Kruppel-like factors, and other sp members come from the family of the transaction that binds GC/GT-rich promoter elements via the 3 zinc fingers of C2H2-type, which are located at the domains of C-terminal. From SP1 to SP4 proteins show the regular expression of more than one gene in normal tissues and tumours. ...
This up and down regulation of SP1 resulted into regulation of hCtr1 expression. Chromatin immune-precipitation assay and SP1 copper regulation are similar and SP1 as self regulated via the intermediation of hCtr1/copper. Safe and Abdelrahim (2005) also noticed the role of SP proteins for the regulation of expression that signalled in lines of cancer cells. Suppression of the P21 by SP3 in cancer cells is similar to acts of SP3 as a repressor of TGF-BRII. Induction of P21 expression is important for cancer cell management for p53 negative cells of cancer. Several chemotherapeutic drugs have been investigated for the induction of P21 expression. Binding of Sp1/Sp3 resulted from antibody super shift and Electrophoresis mobility shift assay demonstrated that de-repression of p21 transaction linked with the increased phosphorylation of Sp1/Sp3 (Xu et al., 2012). Gartel et al., (2000) called the P21 as a negative regulator for cell cycle and found that induction of p21 was expressed in vivo and vitro. Previous studies established that P21 was expressed in the Caco-2 cell lines. The process of P21 in the Caco-2 cells was primarily managed by the factors of SP3 and SP1 by using the two binding sites of Sp1. 2-Materials and Methods The current work required the nuclear extracts. Used the 100-mm culture dishes and 1 mL of PBS for the micro centrifuge tubes. Harvest cells were process at 2500 rpm for 4 minutes at 4 ?C. washed these cells with PBS and also combined the two 100-mm culture dishes for the sample at 4 ?C for 4 minutes with 2500 rpm. Used the Lysis buffer (1ml) and resuspended the cell pellets and allowed to stay for 5 minutes on ...Download file to see next pagesRead More
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