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Gel Electrophoresis

Gel Electrophoresis is a method of separating nucleic acids and peptides using an electric current. The molecules are loaded into a porous gel and an electric current is applied, driving the molecules toward the anode. After the molecules have been separated, various stains and software can be used to visualize and analyze the results.

DTT focuses on changing observable behaviors by using successive approximations of behaviors and shaping new responses. A skill must be broken down into small parts and taught in steps. Reinforcement plays a key role in the success of this treatment. DTT also focuses on being clear and concise in commands and is the overall treatment of choice by most parents and therapists.

History

Gel electrophoresis has remained one of the most powerful analytical tools since its development in the early 1900’s. Prior to modern gel electrophoresis, also known as zone electrophoresis because of the distinct zones of material created, electrophoretic techniques produced poorly separated substances in liquid solutions within a tube. Sample movement was monitored by changes in the resulting shadows (3). This format was described by Tiselius and produced only very partially purified samples of the fastest moving particulates (1). The current standard for electrophoresis, including two-dimensional electrophoresis, which still qualifies as zone electrophoresis, relies on movement and containment of samples within a gel matrix according to their individual charge. Two-dimensional gel electrophoresis provides the highest level of separation attainable through gel electrophoresis and can produce over 1000 unique zones (4).

Originally, single dimension gel electrophoresis was used as a method to separate proteins, however with the discovery of DNA, the focus for most of the mid twentieth century was on isolating DNA strands. This trend continued even after the creation of two-dimensional separation techniques by O’Farrell in 1975 (2). While electrophoresis was utilized for both DNA and proteins after the creation of two-dimensional gel electrophoresis, the use for proteins didn’t increase again until after the human genome was sequenced. It was at this point that the focus of research shifted from genomics towards proteomics. With the increased knowledge from the Human Genome Project, the application of two-dimensional gel electrophoresis for cancer research was ideal. Currently, there are many tools which perform powerful separations including LC, HPLC, UHPLC, CE, and GC, however gel electrophoresis remains one of most common due to its ease of operation, high separation resolution, and low cost.

Underlying Principles

Gel electrophoresis works on the basic physical principles that outline the movement of electrons. Gel electrophoresis is possible because all biological molecules have a net charge, whether it‘s positive, negative, or neutral. This fact is essential in the theory behind electrophoresis and is the basis behind the word itself: “electro” refers to a form of energy and “phoresis” originates from the Greek word “to carry”. Biological molecules, DNA, RNA, or peptides, are loaded into a medium, in the form of a porous mesh gel, and a current is applied. Because the molecules have an inherent charge, those with a negative net charge are pulled via the electric current towards the anode. Because many molecules have the same net charge, but vary in size, more specifically mass, and the moving force is constant, the voltage, molecules move according to their charge to mass ratio: z/m, where z is charge and m is mass. By modifying the gel or chemically modifying the molecules themselves, these ratios can be altered, thereby changing which characteristics the molecules are separated by.

The Child’s Response

Multiple gel types can be used depending on the application. While these gels can vary in the material they’re made of, the composition ratios of said material, and thickness, all gel types work by creating a network of channels for the molecules to travel through. By adjusting specifics of the gel solution, channels are generated in a variety of sizes. Agarose and poly-acrylamide gels are the two most common gel types. Based on the percentage of either, the diameter of the pores can be adjusted, altering the ability of the strands to travel through it. More agarose or more acrylamide create a tighter mesh and restrict the movement of more molecules.

The thickness of the gel can also be altered. This allows for the separation of more or less sample depending on the abundance of the desired molecules. When the sample of interest is very scarce, wider gels hold more sample and therefore increase the concentration of the desired molecule, making it more readily visible after staining. Gel thickness can be altered in conjunction with the desired or available staining options.

Almost all polyacrylamide gels make use of two sections of gel. The first portion of the gel, where the sample will be added, is referred to as a stacking gel. Stacking gels are composed of the same material as the rest of the gel, although they utilize larger pores and different ions to quickly integrate the sample into the gel and maintain the sample within a region between fast moving chloride ions and slow moving glycine ions.

Gradient poly-acrylamide gels are also very common as they are a necessary component of Isoelectric Focusing (IEF). For isoelectric focusing, a pH gradient along the gel must be created in order to alter the charge of molecules as they travel through the gel. In pH gradient polyacrylamide gels, molecules are separated according to their isoelectric point (pI), not according to their mass or size.

Poly-acrylamide gels are subject to a unique problem known as disulfide scrambling however. This happens upon denaturing of the peptides. Disulfide bonds are broken generating sulfide molecules, which can readily form disulfide bonds again. This is problematic because the peptides sulfide ions can form bonds with the acrylamide gels and change the mobility rate.

Gel Analysis

One of the most common follow-up solutions is to stain the gel. Several staining methods are available, again, depending on the purpose of the research. A simple Coomasie Blue stain allows for easy visualization of each spot after separation For samples that might be in extremely low concentrations after separation, more powerful stains such as silver stains can detect peptides in concentrations as low as .1ng/mm2 (Bio-Rad Laboratories, Inc., Hercules, CA). For DNA, the most common stain is ethidium bromide, which is only visible under ultra-violet light and is highly carcinogenic. After staining, several software options exist for analyzing spots. These programs are based on algorithms, which can perform a multitude of calculations. Major components of the programs include background subtraction, spot identification, distance analysis, and the ability to search databases for known molecules presenting similar results. Mass spectrometry (MS) is gaining quickly as the final option as many gel protocols allow for easy sample transfer to MS and mass spectrometers become more advance and more common.

Short Pause Between Consequence and Next Instruction

After giving consequence, the trainer must pause for a short period such as 5-15 seconds before going on to the next trial and repeating the steps.

Pedagogy

Recent research has been based more on how to teach individuals DTT intervention techniques as opposed to if the treatment is effective. Individuals who have been researched in this section include university students, teachers, parents, and direct care staff.

Self-instructional manual and checklists

Self-instructional manuals are one form of teaching DTT to prospective trainers. A study was done at the University of Manitoba in which accuracy increased substantially after giving students a manual to read. [4] Manuals can be effective, especially for people who need a better idea of what DTT is and what is to be expected of them. Also, a trainer can use the manual as a resource to go back to if they have questions.

Checklists are often important in analyzing if DTT intervention is effective or to provide trainers with steps to implementing DTT. Experimenters used a checklist to analyze video recordings of participants and found it was an effective way to gauge accuracy. [5] Trainers can use the steps on the checklist to learn the methods needed to implement DTT and having a checklist to refer to can be really important in promoting generalization.

Video Recording

Video recording is very important in training DTT as well as assessing accuracy. Video recordings are often used to teach the methods of DTT. Video recordings can be very useful in showing a scenario to learners that is representative of an actual session of DTT. Also, it is very cost effective because a video can be shown over and over. Video training is often used in training any job or skill and has shown to be an effective way to teach DTT. Video recording is also very useful to look back at a study and code the data such as how many times a behavior occurred. [6]

Modeling

The use of modeling is important for teaching DTT. In DTT, the learner views the instructor implement part of the treatment and is expected to watch and be able to repeat the methods and steps that they were shown. Modeling is often very effective with learners who do not have an extensive knowledge of DTT because it allows them to see what they need to do. [5] It can also be effective with people who have worked hands on with children with autism because they are often implementing treatments, but do not know the methods they are using by name. [7]

Criticisms

There is a lot of controversy surrounding the use of the word Training in DTT because this term is usually used for non-human teaching such as obedience training with dogs.

References

1. Y. Jin, T. Manabe, Analysis of PEG-fractionated high-molecular-mass proteins in human plasma by non-denaturing micro 2-DE and MALDI-MS PMF. Electrophoresis 2009, 30. 3613-3621, DOI: 10.1002/elps.200900191.

2. P. H. O'Farrell, High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 1975, 250. 4007-4021.

3. P. G. Righetti, Electrophoresis: The march of pennies, the march of dimes. Journal of Chromatography A 2005, 1079. 24-40.

4. O. Vesterberg, Separation of Proteins in Blood Plasma and Serum by Isoelectric Focusing and Two-Dimensional Electrophoresis. National Inst. Occupational Health 1988, 48. 99-101, DOI: doi:10.1080/00365518809168524.

1. Ghezzi, P. M. (2007). Discrete Trials Training. Psychology in Schools, 44 (7), 667-679. doi: 10.1002 /pits.20256

2. Rothstein, A. (2008). The History of Applied Behavior Analysis: It’s not just Discrete Trial Teaching. Retreived from: http://www.autismcentral.ca/research/images/stories/rothstein-aba_article-june_9-08.pdf

3. Behavioral Neurotherapy Clinic. (2009). ABA for Autism. Retrieved from: http://www.autism.net.au/Autism_ABA.htm

4. Thiessen, C., Fazzio, D., Arnal, L., Martin, G. L., Yu, C. T., & Keilback, L. (2009). Evaluation of a Self-Instructional Manual for Conducting Discrete-Trials Teaching With Children With Autism. Behavior Modification, 33 (3) 360-373. doi: 10.1177/0145445508327443

5. Lafasakis, M., & Sturmey, P. (2007). Training Parent Implementation of Discrete-Trial Teaching: Effects on Generalization of Parent Teaching and Child Correct Responding. Journal of Applied Behavior Analysis, 40 (4). 685-689. doi: 10.1901/jaba.2007.685-689

6. Catania, C., Almedia, D., Lui-Constant, B., & Reed, F.D. (2009). Video Modeling to Train Staff to Implement Discrete-Trial Instruction. Journal of Applied Behavior Analysis, 42 (2), 387-392. doi: 10.1901/jaba.2009.42-387

7. Dib, N., & Sturmey, P. (2007). Reducing Student Stereotypy by Improving Teachers’ Implementation of Discrete-trial Training. Journal of Applied Behavior Analysis, 40(2), 339-343. doi: 10.1901/jaba.2007.52-06