SDS-PAGE (1)

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SDS- PAGE
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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Sodium Dodesyl Sulphate Polyacrylamidegel
Electrophoresis

• Separation of Proteins purification of proteins
• According to size mol.wt
• SDS is anionic detergent
• CH3-(CH2)11-O-SO3- - Na

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Procedure

• Samples to be run on SDS-PAGE are first boiled
  with 0.1M ß-mercaptoethanol 1 SDS for 5 min
• ß-mercaptoethanol reduces S-S- bonds and causes
  denaturation
• SDS binds strongly on proteins (Aveg 1SDS 1
  AA) 1.4gm SDS/gm protein
• Protein is denatured and gets ve charge due to
  binding with SDS
• This makes the internal charges negligible and
  the entire protein gets ve charge

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Procedure

• Sample is boiled in a sample buffer
• Sample buffer contains Tracking dye Bromophenol
  blue
• BB allows the passing of proteins through the gel
• All the proteins are vely charged ?anode
• Mobility a mol.wt
• Sieving effect of the gel causes movement of
  smaller size particles easily and vice versa
• Proteins can be visualized by treating the gel
  with Coomassive brilliant blue
• CBB binds to protein but not the gel
• Each band on gel represents a protein
• Smaller proteins are found near the bottom of gel
  and larger proteins at the top

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Applications

• PAGels have very good mol sieving effect than
  starch gels
• Adsorption of proteins is negligible
• Separation of proteins, Nucleic acids
• A combination of Agarose Acrylamide gel is used
  for the separation of High mol wt DNA
• Determines mol wt of proteins
• Purity of mixture of isozymes

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