Paper Electrophoresis

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Paper Electrophoresis

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Principle

• Mainly used for the separation of proteins
• Serum proteins, Hb, LP, Isozymes
• Proteins are charged
• NH2 and COOH groups
• At pI - no movement
• pH gt pI ( protein ? -ve charge ? anode)
• Titration curve

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Apparatus

• 1. Power pack DC 0-500 V or 0-150mA
• 2. Electrophoretic Cell electrodes, Buffer
  reservoirs, transparent insulating cover etc.
• Sample application Spot/streak
• Electrophoretic run Sample equilibrated with
  buffer
• Over heating avoided by cold room
• lt 2hrs
• Paper dried _at_ 1100C
• Detection by comparing with standards
• 1. Fluorescence 2. UV-absorption (260-280nm) 3.
  Staining
• Clinical application Serum proteins etc

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Separation of Aminoacids by Paper EF

• Principle Based on electric charge pH
• Materials required Horizontal EF apparatus,
  Power pack, Ninhydrin, Whatman No.1 filter
  paper(10x2.5), Citrate buffer, PVC tube (5mm
  internal diameter) amino acids (Asp, His, Lys)
• Preparation of Ninhydrin reagent 200mg/100ml
  Acetone

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Preparation of reagents

• Preparation of Standard aminoacids
• 10mg/8ml Ethanol
• Preparation of Citrate Buffer
• 2.101 gm Citric acid ? 100ml H2O ? 0.1M
  Citric acid
• 2.941 gm Sod. Citrate ? 100ml H2O ? 0.1M
  sod.citrate

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procedure

• Power capacity 8 Volts
• Time 3 hours
• 2 compartments are filled with Citrate buffer at
  same level
• Level is maintained by a PVC tube
• About 4 strips of whatman.No.1 (10x2.5cm) taken
• Centre of the filter paper is marked by a pencil
• 4 strips are placed on EF apparatus
• In 3 paper strips ? individual amino acids
• In 4th paper strip ? mixture of amino acids
• Edges of filter paper should touch the buffer
• Buffer moves by capillary action and meet at the
  centre
• Power pack is switched off