Gel Electrophoresis - PowerPoint PPT Presentation

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Gel Electrophoresis

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  1. Gel Electrophoresis

  2. Steps • Breakdown the protein into amino acids • Break amide/peptide bond • Called hydrolysis (opposite of condensation) • Acid or base and heat required • Add protein to gel containing a buffer • Apply a voltage • Amino acids move based on mass and charge • Dye amino acids • Ninhydrin • Measure distance traveled, • Compare to known

  3. Set-up

  4. Why amino acids move • Amino acids separate based on their isoelectric point and molar mass • Isoelectric point: • This is the pH where they net charge of amine and carboxylic acid groups cancel out +NH3-C-COO-

  5. pH of buffer is more basic than isoelectric point, than amino acid will have a negative charge and move toward postive electrode • There are fewer hydrogen atoms around to protonate it NH2-C-COO- • pH of buffer is more acidic than isoelectric point, than amino acid will have a positive charge and move to negative electrode • More H atoms around to protonate +NH3-C-COOH