BIOCHEMICAL CLASSROOM PRACTICE: Electrophoresis OBJECTIVE: electrophoresis with human serum protein (albumin) INTRODUCTION THEORETICAL Gel electrophoresis

is a technique for separating molecules that involves the mi gration of particles in a given gel for applying a potential difference. The mol ecules are separated according to their size because of the smaller mass will mo ve faster than the larger mass. In some cases, the shape of the molecules also i nfluences, as some will be easier to migrate by electrophoresis gel.A is typical ly used to separate proteins and DNA molecules and RNA. Scientifically was intro duced in 1939 by A. Tiselius and EA Kabat in order to separate organic particles . The use of electrophoresis in use in our laboratory has been important to dete rmine hemoglobin and thalassemia, a diagnostic aid for specific diseases, eg.: M ultiple myeloma, and application to lipid, molecular biology and enzymopathies. More sensitive techniques, such as isoelectric focusing allow the separation of fractions with excellent resolution. Agarose gel electrophoresis in this case, is used as the agarose gel electrophor esis. The agarose is a polysaccharide, and form a network that holds the molecul es during migration. Depending on the concentration of agarose, have a differenc e in the gradient separation. To prepare an agarose gel, it is simply a mixture of agarose powder and buffer (TBE). After merging, there is ethidium, which will cause the DNA or RNA "shine" when exposed to UV. When the mixture cool gel will be hard. This hardening is done in an appropriate location, where it will be th e race of the sample. An important detail is the placement of the comb in the ge l during hardening. The comb creates wells that will be used for placement of th e samples. The gel electrophoresis is one of the most widely used methods for an alyzing mixtures of proteins or other macromolecules. The gels used as supports are agarose and polyacrylamide, can be prepared with variable porosity, allowing separation of molecules according to their size, besides its load. Smaller prot eins migrate faster than larger ones, forming a series of defined bands, which c an be visualized by staining with specific. A variant of this technique, known b y the acronym SDS-PAGE, employing a polyacrylamide gel in the presence of the de tergent sodium dodecyl sulfate (SDS). SDS liga3 if the radicals of the hydrophobic proteins, causing their denaturation. This as sociation, with most of the proteins follows the same pattern: one molecule of S DS every two amino acid residues. Moreover, each detergent molecule attaches a n egative charge attached to denatured protein, masking the intrinsic charge of th e molecule natively. The result, whatever protein is the formation of a complex with elongated shape, with the density of negative charges related to the length of the polypeptide chain. This method thus separates proteins according to thei r molecular weight. If the present protein quaternary structure, its subunits ar e denatured and separated by SDS electrophoresis and established the molecular w eight of each of the use of gel electrophoresis, along the different stages of a process of protein purification, except allow its separation, provides addition al information such as: the number of proteins present in the preparation, its m olecular weight and how many subunits are formed. Picture of equipment used for gel electrophoresis 4 • MATERIALS • Cuba electrophoresis Capillaries Becker petri plate (with human blood serum) pap er filter clamp Hairdryer agarose gel • • •

• • • REAGENTS • • • • Tris buffer (9.5) maintains the pH of the agarose gel Acetic acid 5% Black Starc h human blood serum 5 METHODOLOGY 1 - Place 110 mL of buffer in the tank. 2 - Separate the agarose gel of acrylic plate. 3 - Apply serum in agarose gel with microcapillary. 4 - Place the agarose gel in the tub, facing down, matching the (+) side of the gel with (+) in the cell and (-) gel with (-) tub. 5 - Tampa to Cuba, it connects, it app ears that there is bubbles in the corners (indicated passage of electric current ) and leave for 30 minutes. 6 - Turns off the tank. 7 - taken from the agarose g el in the cell, eliminating the excess buffer from the edges with filter paper. 8 - Immerse the gel side up, in 100 mL of dye (black starch) for 15 minutes (wit hout stirring). 9 - taken from the agarose gel dye and place, always turned up i n 100 mL of 5% acetic acid (bleach) with stirring for 1 minute.€10 - Remove the excess bleach from the agarose gel and put it at 60 ° C (use glass or air jet), until it is completely dry. 11 - Place the agarose gel in successive baths of bl each (5% acetic acid) with stirring and after drying using a hairdryer to the ba ckground become transparent again. 12 - To make a qualitative analysis. 6 RESULT * Image Workshops: * Image Internet "Albumin electrophoresis. 7 NOTES • • • The proteins have clusters of positive and negative charges, from th e amino acids that constitute them. Charges are accrued bond or loss of protons Protons (H +) ions are positively charged. Some groups are neutral and to receiv e a proton, becomes positive (the case of group-NH2, which, by binding to the pr oton, is converted to-NH 3 +). Other groups are neutral, but when they lose a pr oton, becomes negative (the case of the-COOH group, which becomes-COO-). These l osses and additions depend on pH when the protein is. • A property of electric c harge in solution is that when a potential difference is applied to the solution - that is, when the poles of a battery are connected to the solution - the posi tive charges are attracted to the negative pole and the negative charges at the positive pole , moving toward them. • The main motive for the movement of charge s in solution is the potential difference. Consequently, the greater the intensi ty of the applied voltage, the greater the speed with which the loads are direct ed to the pole of opposite sign. • • Thus, the higher the load macromolecule, th e greater the speed of electrophoretic migration. The size of the macromolecule also has a decisive influence on their migration velocity. This is because there is a frictional component between the macromolecule, the solvent, the media and other ions in solution which causes the larger the macromolecule, the lower its rate of migration toward the pole of opposite sign. 8 CONCLUSION The lesson was satisfactory, since we can perform the proposed activi ty and it is concluded that: conventional electrophoresis methods are based on t he ability of a gel to separate molecules based on size, under the influence of

an electric field unidirectional. In contrast, we use successive alternating ele ctric fields that force the molecules to change continuously the direction of mi gration. The separation is based, probably in the fact that larger molecules cha nge direction more slowly than smaller ones. However, the precise mechanism of s eparation is not yet fully known, although it is documented in numerous studies. 9 REFERENCES • • • • http://pt.wikipedia.org/wiki/Eletroforese_em_gel http://www.ciencianews. com.br/eletroforeses.htm Marzzoco, and Anita Torres, Bayardo Baptista, Basic Bio chemistry, 2nd edition, New York , 1999, Editora Guanabara Koogan SA http://www. unitau.br/scripts/prppg/biocienc/downloads/comparatecmolec-N22002.pdf 10 11