Electrophoretic Mobility Shift Assay - Lab Report Example

?Electrophoretic Mobility Shift Assay 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. The SP members are essential for metastasis and regulate the expression of more than one gene in cells. Experiment was conducted to perform the EMSA and used P21 promoter that contained 6 sp sites. SP1 was used as an antibody. Sample of nuclear extract was nuclear lystae produced from the colorectal cancer cells with multiple nuclear proteins. 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. It has been found that some of the SP proteins are vital for the metastasis and growth of tumour types as these regulate the expression of cell cycle genes (Safe and Abdelrahim 2005). Specificity Protein 1 controls the (hCtr1) human high-affinity copper transporter in the compressed conditions of the copper stress. Lian et al., (2012) in the research made the observation about SP1; as down regulation of SP1 expression occurred under conditions of copper replete and up regulation of SP1 occurred under conditions of up-regulated. 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 the ice. The process of centrifugation was taken at 4 ?C for 4 minutes with 2500 rpm. Without using the NP-40, the 1 ml of Lysis Buffer was used for washing of nuclear pellets at 4 ?C, for 2500 rpm for 4 minutes. Nuclear pellets were again suspended with 50 ~ 100 µL of the Nuclear Extract Buffer and allowed to stay for 10 minutes on the ice. Process of centrifuge and vortex was again taken at conditions of temperature 4 ?C, rpm 14000 and time 15 minutes. Supernatant was obtained as “nuclear extracts”, and aliquots (10~20 micro liter), freezed it in the liquid nitrogen and shifted to deep freezer at -80 ?C. Bradford assay was used to determine the protein concentration of the nuclear extracts (4 micro Liter). There were three types of buffer required for the current work named as, Lysis buffer, (with and without NP-40), Nuclear extract buffer and 5X Binding Buffer. Current work also required Gel Shift Assay (5% Polyacrylamide gei). The constituents of gel shift assay includes as Distilled water (7.5ml), 10 ml 2X Electrophoresis buffer, 2.5 ml acrylamide bis solution, 200 micro liter of 10% ammonium per-sulphate and 20 µL of TEMED. It was solidified for 1 hour and pre runned for 2 hours at 150 v. Preparation of gel loading sample was taken by using the 14 µL of master mix, 4 mL of Nuclear extracts and 5X binding buffer. At the room temperature above mixture was incubated for 10 minutes. Added 1 mL of the competitors and incubated for the time 15 minutes at the room temperature. Further, 2 mL 32P-labeled oligonucleotide probe was added and incubated for 25 minutes at room temperature. Sample was loaded about 10 mL to the 5% polyacrylamide gel and processed for 3 hours at 150V. Gel was dried and autoradiography was done. After preparation of the reagents the 12x mix, a 5 µL was added to all tubes. Required reagents were added as per requirement and incubated them for 20 minutes. One Liter of 0.5 x TBE was prepared and placed the 5% acrylamide with 0.5 x TBE in the blank plate in the given electrophores unit and also placed a clamp. Inner chamber of the plate was filled with 0.5x TBE. Gel was pre run at the 100V for time 20 minutes. Meanwhile the binding reactions were incubated. During the incubation one more liter of 0.5 x TBE was prepared. In the next step, process of electrophoretic transfer of the binding reactions was carried out to Nylon membrane. Nylon membrane was soaked in the given 0.5 x TBE for 10 minutes. Gel was sandwiched with blotting paper and nylon membrane according to provided instructions. This transfer was taken at 100v for time 30 minutes. Membrane was placed on the bromophenol blue side with a dry paper towel. Buffer was allowed on the surface of membrane in order to absorb the membrane for one minute and never allowed to dry it. In the next step, DNA crosslink was transferred to the membrane. This crosslink was taken at 120ml and stored the dry membrane at the room temperature. Reagents required included were as 40 ml each of blocking and washing buffer, and 30 ml buffer of substrate equilibrium buffer. Other than these reagents, 3 plastic trays were used. In order to dissolve any precipitate, the blocking buffer and wash buffer were warmed at 37-50°C. Membrane was blocked in the 20 ml blocking buffer for time 15 minutes and also shacked it gently. Conjugate/blocking buffer was prepared by the addition of 66.77µl Streptavidin–Horseradish peroxidase to blocking solution 20 ml. Blocking buffer was disposed of and incubated the membrane with a solution of conjugate/blocking for time 15 minutes and also provided shaking. The preparation of 1x wash solution was carried out by dilution of 40 ml of 4x wash buffer to the ultrapure water 120ml. The membrane was transferred to a new container and rinsed it completely with 1x was solution 20 ml. Washing of membrane was carried 4 times for time 5 minutes in each of 20ml 1x wash solution with gentle shaking. About 30 ml substrate equilibrium buffers was added to a new container and incubated it for time 5 minutes and also shacked it. Preparation of the substrate working solution was taken by mixing 6 ml stable peroxide solution with 6 ml of luminal solution. Substrate working solution was poor into the square dish lid and floated down on the membrane face for 5 minutes on the solution without any shaking. Membrane was placed in between the two pieces of the acetate and imaged on the given Biorad imager. 3. Results Figure 1: EMSA image probe used Figure 2: The gel shift assay is made up of 3 key steps of making gel shift assay such as binding reactions, electrophoresis, and probe detection of vitro DNA binding respectively. 4. Discussion: Sp1 consensus Oligo was used as the probe in order to detect the SP1 binding activity in nuclear extract. In order to find the AP2 binding activity, the EBNA Extract was used to find positive control action. Nonspecific complexes either decrease or remain in the presence of specific competitor and addition of any type of the DNA. During the process of gel electrophoresis, the reduced mobility of GSMA relies on the complexity of protein-DNA. During the process of electrophoresis and its dissociation the detection of complexes is prevented, and slow dissociation results into the underestimation of the binding density. Complexes remain stable in the gel as compared to free solution. It has been seen that buffers are compatible with the electrophoresis. Pull down assay was used for the selective extraction of a complex protein DNA from sample. Gel was imaged on the glass plates. By removing the gel from glass, avoid to tear the gel. The study of Mansilla and Portugal (2008) also confirmed the results of the current research work about development of the anti tumour agents. It has been also found more potent inhibitor for the transcription initation from promotor having the SP1 binding site, and also in vitro cell lines. From the research work of Koutsodontis, Moustakas and Kardassis (2002) the SP1 and SP3 members were binded to the 6 GC-rich motifs in the proximal promotor from the human cell cycle. Current work showed that SP1 and SP3 were bound to a high afficnity elemnts. The results suggested that functional interaction of the SP1 family members were bound to particular elemnts. It was also found that proximal promoter factors bounded to the elemnst governed P21 hepatic activity under the inducible or basal conditions. By using the SP1 and SP3 the dependency of genes activiation was investigated on the SP family. Both SP1 and SP3 binded towards the DC-rich sequence and were found to be cooperative. The study of Smith and Delbary-Gossart (2001) provided another evidence on the binding of the DNA proteins towards the genes of a regulstory region. As, in case of the current study, the information about transaction factors at the given time was as essential as as it generated respond to the environment. It was seen that P21 protected the cells from severe genotoxic stress and prevented the cycling of most damaged cells. This role of P21 was also studied by Ju, Chouhdary and Rudolph (2007) in their work on the role of p21 for the aging of stem cells. Conclusions: Rapid method of Electrophoretic Mobility Shift Assay for detection of DNA-binding proteins has been discussed. Role of SP1 as anti body and P21 in cancer cells has been briefly described. Current work used the buffer solution and other essential reagents for processing of the experiment. Experiment and methods part included the preparation of nuclear extracts, gel shift assay and buffers. Role of P21 was observed as management of the cell cycle and particularly in sample cells of the current study. Role of SP1 was seen an antibody in the cells. References: Gartel., A., Goufman, E., Najmabadi, F., and Tyner, A. (2000). “Sp1 and Sp3 activate p21 (WAF1/CIP1) gene transcription in the Caco-2 colon adenocarcinoma cell line”, Oncogene 19, 5182 -5188. Ju, Z., Chouhdary, A.R., and Rudolph, KL. (2007). “A dual role of p21 in stem cell aging”, Ann N Y Acad Sci. (1100)333-44. Koutsodontis, G., Moustakas, A., and Kardassis, D. (2002). “The Role of Sp1 Family Members, the Proximal GC-Rich Motifs, and the Upstream Enhancer Region in the Regulation of the Human Cell Cycle Inhibitor p21WAF-1/Cip1 Gene Promoter”, Biochemistry, 41 (42), pp 12771–12784. Liang ZD, Tsai WB, Lee MY, Savaraj N, and Kuo MT. (2012). “Specificity protein 1 (sp1) oscillation is involved in copper homeostasis maintenance by regulating human high-affinity copper transporter 1 expression’, Mol Pharmacol. 81(3) p.p.455-64. Mansilla, S., and Portugal, J. (2008). “Sp1 transcription factor as a target for anthracyclines: Effects on gene transcription”, Biochimie Volume 90, Issue 7, July 2008, Pages 976–987. Smith, M., and Delbary-Gossart, S. (2001). “Electrophoretic Mobility Shift Assay (EMSA)”, Methods in Molecular Medicine Volume 50, 2001, pp 249-257 Safe, S., and Abdelrahim, M. (2005). Sp transcription factor family and its role in cancer. European Journal of Cancer 41 2438–2448 Xu, W., Zhu, Q., Wu, Z., Guo, H., Wu, F., Mashausi, D., Zheng, C., and Li., D. (2012). “A novel evolutionarily conserved element is a general transcriptional repressor of p21WAF1/CIP1”, American Association for Cancer Research. Read More