Expression and Purification of recombinant Green Fluorescent Protein

Expression and Purification of recombinant Green Fluorescent Protein ABSTRACT: The purpose of this experiment was to determine if a His-6 tagged recombinant form of Green Fluorescent Protein could be expressed in a pRSETA vector of E. Coli. This was determined through multiple procedures beginning with purifying the sample with Ni +2 agarose chromatography which showcased the relative fluorescent activity of the samples, which elution sample two (E2) had approximately 100,592.2 RFU/mg . The yield of total protein was found by use of a Bradford Assay and a standard curve. The purity of the GFP was determined by comparing the intensity of bands that appeared at around 31.4 kDa (the molecular weight of rGFP) to a molecular weight ladder on an SDS-PAGE gel. The Western Blot test, utilizing a nitrocellulous membrane, confirmed the expression of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel which E3 had an estimated purity of 0.4, indicating a yield of approximately 29.4 ug of rGFP for the third Elution (E3) after a total p rotein amount of 49 ug was extrapolated from the Bradford assay. INTRODUCTION: The Green Fluorescent Protein was first isolated from the Aequorea Victoria jellyfish and studied by Osamu Shimomura. In other organisms other than this specific jellyfish, there are fluorescent proteins with more color varieties than just green (Ward). GFP is able to fluoresce due to the formation of a chromophore in its center that resides in a stable beta barrel structure. The interactions between Ser64 and Gly67 on an alpha strand form a five member ring structure and its this structure that contains properties for fluorescence. When ultraviolet light is absorbed at 395nm, an emission wavelength of 510nm is seen as fluorescent green light. The excitation wavelength of 395nm excites the electrons within the protein and raises the energy of the protein. As the protein loses energy, it emits the energy at a wavelength of 510nm. Wild type GFP does not have immensely visible fluorescence, therefore a genetically modified form of GFP, GFPuv, was used in order to better observe fluoresc ent activity. For rGFP to be fully expressed, GFPuvs open reading frame was pRSETA vector, which was cut by a restriction enzyme at sites that complimented the start and stop codons of the His-6 tag sequence. A T7 promoter and Xpress Epitope were also genetically added into the vector with the purpose of simplifying purification. T7 polymerase minds to the T7 promoter and activates the production of GFP, while the Xpress Epitope allows for the binding of a primary antibody. The binding of a primary antibody allows for the binding of a secondary antibody which contains horseradish peroxide, producing bands on the nitrocellulose membrane. (2 pg 351) Histidine-6 tags primary purpose is to aid in the purification of rGFP proteins in affinity chromatography. Histidine has an affinity to bind with transition metal ions, and is why a Nickel+2 agarose chromatography was chosen. When a sample is passed through the Nickel+2 agarose column, the His-6 tags fused with rGFP protein will bind to the column and any proteins not of interest or containments will pass through the column. To elute the bound rGFP proteins from the column, elution buffer containing Imidazole is used and competes with the histidine residues for the binding sties to the Nickel+2 agarose column due to its higher affinity for them. Elutions from the column therefore contain rGFP. (2 pg 359) The purpose of this experiment was to see if a His-6 tagged recombinant form of GFP, from the E. coli strain BL21pLysS, could be expressed and then purified using Ni+2 agarose affinity chromatography technology. If this experiment is successful, an inexpensive and relatively simple method to measure gene expression and locate and track proteins may be at hand. MATERIALS AND METHODS: Bacterial Expression Grow two bacterial cultures: G, containing BL21, DE3, pLysS, and the plasmid pRSETA-GFPuv with the GFP sequence, and V, which is identical to G, but it does not have the GFP sequence. Incubate both cultures at about 37Â °C until OD600 equals 0.5. Transfer 1ml of each culture into separate centrifuge tubes and centrifuge to obtain a pellet. The supernatant from the tubes will be discarded and then label the tubes V0 and G0 and stored at -20Â °C. Induce the rest of the culture with IPTG and allow to grow for 3 hours. After the 3 hours, collect 1ml of each culture and centrifuge. Label the tubes V3 and G3 and were store at -20Â °C. Collect 15 ml of G and centrifuge, and label it as G3-15ml and store at -20Â °C. (1 pg 105) Preparation of Crude Extract After the slow freeze process, add 1 ml of breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) to the frozen bacterial pellet G3-15ml, and pipette up and down until completely dissolved. Then transfer the contents to a centrifuge tube and vortex for 5 minutes and place in a 37Â °C water bath for 10 minutes. Afterwards, incubate in dry air at 37Â °C. Centrifuge the mixture at 4Â °C and transfer the supernatant into a clean centrifuge tube and take a small sample to label as GCE. The remaining supernatant will go through the Nickel+2 agarose column. (1 pg 110) Ni+2 NTA Chromatography Pack a 3ml plastic syringe with a small amount of glass wool to prepare the Ni+2 agarose column. After that, pipet 1 ml of breaking buffer into the syringe to remove any air bubbles. As the breaking buffer is going through the syringe, pour some breaking buffer into the leur-lock and then screw it onto the syringe. Add 1ml of 50% Ni+2 agarose into the column and then open the leur-lock to pack the agarose matrix in the colum. Add 5 ml of breaking buffer subsequently into the column to push the ethanol of the Ni+2 agarose through the column. Then close the leur-lock and apply the crude extract to the column and allow 5-10 minutes for the histidine tags on the rGFP to bind with the Ni+2 agarose beads. Open the leur-lock and collect the non-crude extract in a centrifuge tube labeled W1 for the first wash. Add 0.5ml aliquots of breaking buffer to the column and collect the washes in tubes W2-W10. Add 10 increments of 0.5ml of elution buffer and collect in tubes E1-E10. Then store your GC E, W1-10, and E1-10 samples at -20Â °C. (1 pg 111-2) Bradford Assay Analysis To create a Bradford assay, mix protein sample with water, and then add Bradford reagent dye. Vortex the mixture and then transfer it to the wells of a microtiter dish, so that the absorbance of your protein sample can be determined at 595nm using a spectrophotometer. To determine the amount of total protein present in the sample volume, you must create a Bradford standard curve using 0, 2.5, 5, 10, 15, and 20 ug of BSA and the absorbance values observed to determine a best-fit line. Perform the assay in singlicate using samples W1-W6 and E1-E6 to determine if the microplate data falls within the standard curve. Once you have determined what volume of sample to use, repeat the Bradford assays two more times for each sample. Use the standard curve and experimental absorbance values to extrapolate the total protein present in the volume of sample you use. (1 pg 124-6) SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions Create a 12% Resolving Gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x resolving buffer [0.75M Tris pH8.8, 0.4%SDS]. Pour the resolving gel into a gel electrophoresis set up and overlay with some water. Allow the resolving gel to polymerize and proceed to making a 5% Stacking gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x stacking buffer. Pour the stacking gel on top of the resolving gel and immediately insert a comb to form the wells. Prepare your loading samples with 4x sample loading buffer and then vortex, boil, and centrifuge. Do this for the G0, G3, GCE, W2, W3, E2, and E3 samples (or fractions that contained the most rGFP fluorescence). (1pg 111-2) After the gel has solidified, transfer the apparatus into the electrophoresis tank and load the electrophoresis buffer into the tank. Then load your samples into lanes one through seven respectively along with a marking ladder to compare the distances the samples traveled after electrophoresis. Electrophorese at 200V for approximately 45 minutes and then stain your gel with Coomassie Blue dye. To remove the stain that has not been absorbed by proteins, the gel is soaked in dilute acid and methanol. Western Blot Develop an SDS-PAGE gel as previously described using your sample fractions and transfer the proteins onto nitrocellulose transfer membrane using two locking cassette lids with sponges and filter paper encompassing the gel and nitrocellulose. Remove the nitrocellulose and stain the membrane with Ponceau S stain, and allow to incubate for approximately 2 minutes. Once stained, wash the membrane with nanopure water until bands are visible on the membrane. Next, perform the blocking step by placing the membrane in a container containing 5% non-fat dry milk/TBS solution and incubate on a shaking platform. Pour out the blocking solution and add 0.05% Tween 20/TBS solution and then allow it to incubate on a shaking platform, and then pour out the solution. Repeat this wash step two more times. Add mouse IgG anti-Xpress epitope MAb and allow incubate on a shaking platform this is the primary probe step. Then repeat the wash step as mentioned previously three more times, and add Sheep IgG a nti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution (the secondary probe). Allow the membrane to incubate, and then perform the wash step twice. For the final wash, use only TBS, and then add TMB substrate solution and incubate until appropriate banding is shown. Stop the reaction with distilled water and scan or photograph the membrane as the results may fade with time. RESULTS: In the specific strain of E. coli, BL21pLysS, pRSET-GFP represses the lac promoter which is part of the operon needed for T7 polymerase activity. T7 polymerase binds to the T7 promotes of pRSET-GFP and activates the His-6 tag that expresses the fluorescence of GFP. Even though some GFP is produced this way, it is experimentally not enough, so IPTG is added to repress the repressor so an increase of GFP production can occur. V0, V3, G0, and G3 were all bacterial cultures of E. coli with specific distinctions between all of them. V0 and G0 were collected from bacterial cultures that were not yet induced, but G0 contained the GFP sequence while V0 did not. V3 and G3 were the V0 and G0 samples after induction had occurred for 3 hours, and again G0 contained the GFP sequence while V0 did not. W1-W10 were washes collected from the Ni+2 agarose column using the breaking buffer whereas E1-E10 were washes collected using elution buffer. Figure 2. SDS-PAGE/Coomassie Blue stained gel analysis of rGFP SDS-PAGE gel, which was used to analyze our purification of rGFP from a crude extract, and the corresponding molecular weight. This gel was made with a 12% resolving gel and a 5% stacking gel. Wildtype rGFP has a molecular weight for 27kDa. The ladder has kDa rungs of 97.4, 66.2, 45.0, 31, 21.5, and 14.4. The band for rGFP has been indicated on the figure. The different samples were taken during our purification. The highest protein containing washes and elutions were used in the SDS-PAGE gel. G0 represents a sample containing rGFP prior to induction. G3 represents a sample containing rGFP after 3 hours of induction. GCE represents the GFP crude extract after the slow-freeze/quick-thaw process. Figure 3. Western Blot of rGFP We used the Western Blot to detect, and more importantly, quantify proteins that react with antibodies. The Ponceau S stain was performed to highlight the existence of our proteins successfully being transferred from the SDS-PAGE gel. We then performed a blocking step with 5% non-fat dry milk/TBS solution followed by washes with 0.05% Tween20/TBS. The probe first used was mouse IgG anti-Xpress epitope MAb solution, which we followed with washes of Tween20/TBS solution. The second probe used was sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution, which we followed with two washes of Tween 20/TBS solution with the third wash being pure TBS. Then, TMB solution was added to develop the nitrocellulose membrane and this reaction was stopped by pouring distilled water over the membrane. Above are the 8 lanes, G0, G3, GCE, W2, W3, E3, E4, and a known molecular weight ladder, respectively. CONCLUSION/DISCUSSION: A great amount of research has been put into the field of monitoring gene expression and there are currently many research laboratories that use labeled antibodies and other various means to do so. However, this new recombination technology, a vector can be created to provide a simpler and less expensive approach that can even be performed in vivo. The purpose of this experiment was fulfilled as His-6 tagged rGFP from a particular E. coli strain was successfully expressed, purified, qualified, and quantified by means of Ni+2 agarose chromatography. GFP was initially expressed as a histidine tagged protein that was inserted in E. coli and subsequently a crude extract of this protein was isolated. Throughout the experiment, we were able to inspect and measure the fluorescing activity of our elutions with the use of handheld UV lights. GFP was successfully expressed because of the fluorescent activity that was emitted when shown under UV light. This rGFP was purified using a Ni+2 agarose column which allowed for the binding of the His-6 tagged rGFP to the Ni+2 agarose beads in the column, and subsequently let all other proteins not of interest and containments to be collected in washes. Then, rGFP proteins were eluted by passing elution buffer containing Imidazole through the column because Imidazole has a higher affinity for the Ni+2 agarose beads than does Histidine. Therefore, one could expect that most if not all of the rGFP would be found in the elution washes and would present higher fluorescing activity than the washes with breaking buffer. Figure 1 clearly supports this argument. From referencing Figure 1, the most rGFP was found in E2 because it exhibited the highest fluorescent activity in RFUs. Using a standard curve based off of known quantities of mass of a certain protein, one is able to extrapolate the amount of protein present in ones sample. This can be used to estimate the amount of rGFP activity. For instance, E2s specific activity was found to be approximately 100,592.2 RFU/mg. W1-W6 had the least amount of fluorescing activity because the washes were done prior to the addition of Imidazole through the column. This is expected as Imidazole would flush out rGFP in the elutions, which would produce the highest GFP fluorescing activity. Percent purity of the rGFP samples were approximated using the SDS-PAGE gel with Coomassie blue staining to allow visible bands to appear which were used for comparisons. The molecular mass from each individual sample was determined by comparing the bands with the bands of the known weight ladder. The percent purity of each individual band was determined by comparing the bands against each other. The molecular weight of wild type rGFP is 27 kDa, and the molecular weight of rGFP for mutant rGFP used for this experiment was approximately 31.4 kDa with the 40 extra amino acids that were added taken into account. For example, E3 percentage of purity was found to be .4. The amount of protein yielded was 29.4 ug of rGFP for the third Elution fraction (E3) after a total protein amount of 49 ug The Western Blotting technique detected and quantified proteins that reacted with a specific antibody. In our case, it verified rGFP was expressed and whether or not it fell in the range of the expected molecular weight of 31.51 kDa. Lanes GCE, G0, G3, and E3 all contained a bright band around 31.4 kDa, indicating that rGFP was existent. This result corresponds to the expected of E3 having the one of the highest rGFP activity. The fact I had used E3 for the SDS-PAGE and Western Blotting skewed my results, but E2 and E3 had the highest activity for me. Even with my error, GFP was effectively expressed and purified and its finding is important to science because it can be used to track cancer causing cells one day to help figure out where problem spots begin and can help eliminate the issue before it escalates indicating that fluorescent proteins might eventually be clinically useful in cancer patients (Hoffman). This can occur since GFP can successfully be expressed in other organisms as this experiment through the use of E.coli, humans cells can certainly be tagged and label the tumour before treatment and then monitor for fluorescence after treatment to identify possible recurrence or metastases (Hoffman). The same can be said with heart disease and other health related problems. The future of GFP remains limitless when scientists can unlock its full medical potential. REFERENCE/BIBLIOGRAPHY (1) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Manual. The University of Texas at Dallas. Richardson, TX. (2) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Lecture Notes. The University of Texas at Dallas. Richardson, TX. (3) Ward, William. History of GFP and GFP Antibodies. Brighter Ideas. 2009. 26 Oct, 2010. http://www.brighterideasinc.com/proteins-antibodies/history-of-gfp-and-gfp-antibodies/ (4) Ninfa, Alexander J. and David P. Ballou (1998). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. (5) Hoffman, Robert M. Uses of Fluorescent Proteins to Visualize Cancer In Vivo: Can Fluorescent Proteins be Used in Humans? 2005. 28 Oct. 2010. http://www.medscape.com/viewarticle/513975_6

Library system Essay

Chapter I Introduction The world of Information Technology transformed and made the life of human beings easier through the innovation of different machines and software applications. Faster and reliable sources have been offered by search engines which are all over the web. But even though this is the case, many schools and universities are still requiring their students to use library materials in their researches and school works. From the statement of purpose, the compute’rized library system: meeting information needs of the people of Saint Michael College of Caraga, Nasipit, Agusan del Norte. Our proposed system, the Saint Michael College of Caraga Library Management System, is a system wherein there is no need of manual library transactions. This will carry out different processes such as searching books, keep records of the books, borrowing of books and return of books. Hence, at present, Saint Michael College of Caraga is maintaining the School Library in a manual basis. Handling bulky records in a manual method is very difficult. And the process of updating could not be done easily and  accurately. Likewise, a manual procedure of handling bulky record is very slow and is prone to manual errors. Consequently, it is proposed that Saint Michael College of Caraga shall maintain an electronic data base management system (DBMS) for the purpose of maintaining the records of the School Library for easy, fast and accurate processing and maintenance of these records that will enhance management decisions and for the improvement of its services to all the stakeholders of Saint Michael College of Caraga. Library is regarded as the brain of any institute; many institutes understand the importance of the library to the growth of the institute and their esteem users (students). Library Management System of Saint Michael College of Caraga supports the general requirement of the library like acquisition, cataloguing, circulation that offers many flexible and convenient features, allowing librarians and library users to maximize time and efficiency. Library System gives the all detailed information about students, staff and books. It will track on the how many books available in library and books issued to the students. It shows popular book among the students. It will provide book lost in library. It keeps the record of the suppliers and book binders. It generates MIS reports for management. Our software is customizable for any library requirement. In this chapter we take opportunity to consider changes in the library services, not in terms of minor, short term change in equilibrium of the sort just noted above, but in longer term. There has been continuing concern about the future of the libraries. Given the extent to which the provision used and the libraries are influenced by the social environment, and given possibilities by the use of new information technology; it would be unreasonable to expect libraries to remain static. But if not, what would be the nature of the change? The issue is not whether there is a change but what will be the change. If we are to make a claim to understand the nature of library services, the surely we ought to have notions, some forecasts, about how library system might change. Purpose and Description In setting up a library, one aspect that should be considered, what are the resources the library has and what are the ways to provide a better service to the students? Well, one of the easiest ways is having a system to  organize all the transactions in the library. A library system is software that will handle basic and systematic organization of function in the library. The system would provide basic set of features to add/update student’s information, add/update books information, search for books and manage check-in/check-out processes. In this application we can maintain the records of students and books and enable to determine how many books are issued and likewise determine the available books in the library. The proposed library system will greatly improve the efficiency of the school library. This study has the following hypothesis: The profiles of the respondents taken are their names, Student ID Number, Year and Course, and Title and Author of the Book Borrowed. The manual system is very time consuming, inconvenient when it comes to recording, organizing and retrieving borrower’s record in the log book. There are few solutions but many a times, we â€Å"making them works† by finding â€Å"ways to work around† system inadequacies. Moreover we have limited staff resources, these â€Å"work around† waste time, effort, and skills that should be spent on user services. solutions do not keep up with the technological changes and hence prove to be time wasters. Library Management system is a small footprint software suitable for personal /individual Libraries. You can store the information about the books and other material and control the movement of the same. Silent Features †¢ Control the movement of books and other material and avoid losing the same. †¢ Search if you have a specific book in your collection based on t he title, author etc. †¢ Print the spine labels for the book. †¢ Find what a specific person has borrowed from you. Objectives The main objective of the application is to automate the existing system of manually maintain the records of the Book Issue, Book Return from the student, Stock Maintenance, and Book Search to be computerized. And to develop a database which stores user details and book details, give reliable search facility for the user, create an easy to understand user friendly environment. So the Book Issue, Return, Searching will be faster. This  application can be used by any Library to automate the process of manually maintaining the records related to the subject of maintaining the stock and Book Issues. The general objective of this study is to design and develop a library system that will serve as a proposal to help librarians save time with the automation of its daily operation. Specific Objectives: To computerize records keeping of books; To allow librarians to retrieve complete information of the book and its borrowers. To check the availability of the books and penalties. To search, issue and return of books. It can only be utilized by the librarian Scope and Limitation The study only focuses on the Information Technology Library Management System of Saint Michael College of Caraga. Without computers, as some libraries are, all of them are dependent on paper work. When compared to computerized systems, data backup and data retrieval systems are inefficient and labor intensive. Users of a paper based system become almost wholly reliant on the librarians, for all levels of service, whether they are enquiries about existing books, inter-library loans, or the availability of books. There is additionally no real involvement of a user in the entire process. The transactions that the system accommodates are the attendance of every student that enter the library, book registration and deletion, updating book information and searching thesis references, Borrowing of books, and keeping the record of transaction. Review of Related Literature This chapter contains the relevant outline of literatures related to the study of Library system. This study composed of programming languages, Database and Graphical User Interface (GUI) that is used in the system. This system would be used by members who may be students or professors of that University to check the availability of the books and borrow the books using automated device, and by the librarian to update the databases. The purpose of this document is to analyze and elaborate on the high-level needs and  features of the Library Management System. It focuses on the capabilities and facilities provided by a Library. The details of what all are the needs of the Library Management System and if it fulfils these needs are detailed in the use-case and supplementary specifications. Automated book monitoring system helps to reduce the effect of entering the wrong quantity and the amount of staff time devoted to repetitive activities. Related Studies According to ACRL (1994) suggested that institution should be prepared to utilized new technologies for accessing information as they are developed. This is why operating the library operations is quite a welcome idea. According to Jeff Kaplan (05/08/2007 A new generation of automated network management software and services is helping in-house staff address this challenge. These let network professionals establish regular patch-management procedures to safeguard against escalating security threats; and create system monitoring routines to identify load imbalances, which could cause service disruptions or performance problems. They also discover, inventory and track assets to make sure that hardware and software licenses are up-to-date and that problems can be resolved faster. According to Voustin Sweere (Aug.2001) Nowadays every book keeping system used in practice is automated. Most book keeping software an integrated information system are based on database. In this paper, we develop a conceptual book keeping model which is not based on manual techniques, but which is applicable in database environment. Technical Background In this chapter, it provides an overview of all the overall system design, features and functionalities. The system is design to implement a computerize library management system in Saint Michael College of Caraga. This system consists of one user, the administrator. The administrator can  access the system by having first the login security which they have to input his/her user name and password. Once the inputted username and password did not match, the required/registered information it displays â€Å"Invalid Username and Password†. If the username and password match the registered information, the next form will display depending on the role of the administrator.

The Student Loan Market Is A Growing Component Of The U.s....

The student loan market is a growing component of the U.S. economy. Student loans are the second largest source of household debt. Assisting borrowers with the loan repayment process, like finding the right repayment plan and avoiding default, is an essential component of preventing further drag on the economy. Further, illuminating practices unfriendly to consumers and improving student loan servicing can potentially decrease the default rate and expedite the repayment process. To end these practices, we recommend that the CFPB use its authority to promulgate regulations and implement new student loan servicing protections for borrowers in the following ways: 1. Require student loan servicers to do their part to counsel borrowers about their repayment options using consistent, industry-wide policies 2. Require servicers to send emails to borrowers recapping each interaction or transaction 3. Require student loan servicers to follow borrowers’ payment instructions 4. Issue guidance for student loan servicers to follow when performing account transfers Many borrowers try their best to repay their student loans but encounter unfair policies designed to extend their repayment periods. In April, we conducted an online survey to explore the â€Å"borrower experience† during repayment. We learned that borrowers are doing all or the right things: they borrowed money to get an education necessary to open doors to opportunities. When they entered repayment, however, the frustration,Show MoreRelatedThe Duration Of Unemployment As Provided By The Bureau Of Labor Statistics1704 Words   |  7 Pagessignificantly to 33.7 (BLS 2015). The average hours in 2006 before the Great Recession were also around 34.5 hours, so I would say the labor market has done a good job recovering from the recession (BLS 2015). Even two years ago it seems that we had not fully recovered, when the average weekly hours worked hovered around 34.4 (BLS 2015). 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A Lifetime of Damage Essay - 2397 Words

Overview The purpose of this research report is to review the practice of corporal punishment in the home and the lasting harmful effects on the children involved. The research is based on the opinions and finding of professionals in mental health and child development and offers little to no support for the use of physical punishment. This research outlines several areas of a child’s life that are acutely affected by the violent form of punishment called â€Å"spanking†. Within our community I have seen first hand that children who are subjected to such punishment suffer emotional as well as physical harm. Loss of trust, confusion, damaged relationships; aggression, humiliation, depression as well as death occur in children disciplined†¦show more content†¦Even later in life as these children grew into young adults the lasting effects of physical punishment could still be seen. The association of love with violence that is learned through corporal punishment causes ant isocial and violent behaviour later in life (Straus, 1994; Greven, 1991; Flynn, 1999). Children and spouses of individuals who were spanked have a much higher risk of being battered (Straus, 1994). According to a 1985 National Family Violence Survey published by Murray Straus, the risk of drug and alcohol abuse is also much higher in the population of children whose parents spanked them. The use of drugs and alcohol may create an escape for those individuals looking for a diversion from their traumatic memories of perceived rejection as children (Straus 1994). A child who is spanked not only absorbs the blows, but the message the blows convey, â€Å"You’re worthless, I reject you!† Murray Straus, author of Beating the Devil Out of Them, writes, â€Å"No one of us can show that anything bad happens if we do not hit children† (Straus, 2000, p.1). 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