Foundations of Biology

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					   SDS-PAGE
Sodium Dodecyl Sulfate -
   Polyacrylamid Gel
    Electrophoresis
     Sodium Dodecyl Sulfate
SDS is a common ingredient in detergents
Other names for SDS include laurel sulfate and
sodium laurel sulfate
As a detergent SDS destroys protein secondary,
tertiary and quaternary structure
This makes proteins rod shaped
SDS also sticks to proteins in a ratio of
approximately 1.4 g of SDS for each gram of
protein
Negative charge on the sulfate groups of SDS
mask any charge on the protein
                               SDS
                   Sodium Dodecyl Sulfate
                       C12H25NaO4S

   HHHHHHHHHHHH O
H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-N
   HHHHHHHHHHHH O
              Non-polar                            Polar
            Hydrophobic tail                  Hydrophilic head



Because it is amphipathic, SDS is a potent detergent
SDS and Proteins

                   SDS




     Protein
           SDS and Proteins
SDS nonpolar chains arrange themselves on proteins and
destroy secondary tertiary and quarternary structrure
Thus shape is no longer an issue as the protein SDS
complex becomes rod shaped


In aqueous solutions, SDS polarizes releasing Na+ and
retaining a negative charge on the sulfate head
So much SDS binds to proteins that the negative charge
on the SDS drowns out any net charge on protein side
chains
In the presence of SDS all proteins have uniform shape
and charge per unit length
        Polyacrylamide Gels
Polyacrilamide is a polymer made of acrylamide
(C3H5NO) and bis-acrilamide (N,N’-methylene-
bis-acrylamide C7H10N2O2)

                         CH2 CH    Acrylamide
        O
                          O   C   NH2
        C   NH2
                                  CH2
   CH2 CH
                          O   C   NH2
                                   Acrylamide
   Acrylamide            CH2 CH

                         bis-Acrylamide
        Polyacrylamide Gels
Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate



         O           O

         C   NH2     C    NH2
    CH2 CH       CH2 CH

             SO4-.
           Polyacrylamide Gels
1. Acrylamide polymerizes in the presence of free
  radicals typically supplied by ammonium persulfate
  2.TMED (N,N,N’,N’-tetramethylethylenediamine)
  serves as a catalyst in the reaction
             O          O            O         O

             C   NH2    C      NH2   C   NH2   C   NH2
        CH2 CH    CH2 CH        CH2 CH    CH2 CH



                       SO4-.
          Polyacrylamide Gels
bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
     O                      O         O

     C    NH2               C   NH2   C   NH2
 CH2 CH    CH2 CH    CH2 CH      CH2 CH

            O   C   NH2
                    CH2     O         O         O

            O   C   NH2     C   NH2   C   NH2   C   NH2
           CH2 CH    CH2 CH      CH2 CH    CH2 CH
           bis-Acrylamide
         Polyacrylamide Gels
bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
          Polyacrylamide Gels
Pore size in gels can be varied by varying the ratio
of acrylamide to bis-acrylamide
Protein separations typically use a 29:1 or 37.5:1
acrylamide to bis ratio




                                   Lots of bis-acrylamide
                                    Little bis-acrylamide
            SDS-PAGE
1   2   3




                                    3
                    Addition of SDS
                          2
                1   Protein becomes rod-
                    shaped with uniform
                    charge distribution

				
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posted:9/5/2012
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