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PAPER ELECTROPHORESIS

BIOCHEMISTRY

mprasadnaidu
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PAPER ELECTROPHORESIS

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  1. Paper Electrophoresis M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

  2. 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 > pI ( protein  -ve charge  anode) • Titration curve

  3. 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 • < 2hrs • Paper dried @ 1100C • Detection: by comparing with standards • 1. Fluorescence 2. UV-absorption (260-280nm) 3. Staining • Clinical application: Serum proteins etc

  4. 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

  5. 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

  6. 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

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