Comparación de métodos de extracción de proteínas de cerebro y linfocitos de rata
Comparison of protein extraction methods from brain and lymphocytes of rat
Resumen
En este trabajo se compararon tres técnicas de extracción de proteínas actualmente empleadas en proteómica, para determinar la más eficiente para realizar electroforesis bidimensional (2-DE) en tejido cerebral y linfocitos de sangre periférica de rata. Los métodos utilizados fueron el uso directo de solución de lisis, el método TCA/acetona-DTT y el método TCA/acetona-fenol. Una vez que se realizó la extracción, se separaron las proteínas por medio de electroforesis en geles de poliacrilamida en condiciones desnaturalizantes (SDS-PAGE) y 2-DE, con el objetivo de seleccionar cuál de ellos brindó un mayor rendimiento en la cantidad de proteínas totales, así como en el número de bandas bien definidas y manchas bien enfocadas en los geles 2-DE, tanto para cerebro como para linfocitos. Al comparar el perfil proteico, en cerebro se detectaron 13 ± 0; 15 ± 1 y 12 ± 1 bandas bien definidas mediante los métodos de TCA/ acetona-DTT, TCA/acetona-fenol y solución de lisis, respectivamente. En linfocitos, se encontraron 19 ± 1.20 ± 0 y 19 ± 1 bandas, respectivamente. Con respecto al proteoma, tanto en cerebro como en linfocitos se encontró mayor número de manchas proteicas consistentes y bien enfocadas con el método de TCA/acetona-DTT. Estos resultados mostraron que el mejor método de extracción de proteínas para su uso en la 2-DE correspondió al de TCA/acetona-DTT, siendo además más rápido y sencillo de realizar que el método de TCA/acetona-fenol.
Citas
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