Paper electrophoresis

about paper electrophoresis

1. 1 PAPER ELECTROPHORESIS Presented by :- Pradnya Shinde. M.Pharm –I year Dept. P’ceutics R.C.P . kasegaon.

2. INTRODUCTION The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation. The support medium is a filter paper . The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution , through which electric current is passed. Frequently used in isolating proteins, amino acids and oligopeptides. 2

3. PRINCIPLE- “ The charge carried by a molecule depends on the pH of the medium. Electrophoresis at low voltage is not usually to separate low molecular weight compounds because of dif fusion, but it is easier to illustrate the relationship between charge and pH with amino acids than with proteins (or) other macromolecules.” 3

4. INSTRUMENTATION The equipment required for electrophoresis consists basically of two items, a POWER PACK and an ELECTROPHORETIC CELL . Power pack : provides a stabilized direct current and has controls for both voltage & current output, which have an output of 0 to 500V and 0 to 150mA are available . The Electrophoretic cell: contains the electrodes,buffer reservoirs , a support for paper and a supporting transparent insulating cover. The electrodes are usually made of platinum . 4

5. The two arrangements of the filter strips are commonly used . The horizontal & vertical arrangements . Both the arrangements are equally viable & the choice usually depends upon personal preferences. Filter Paper : Paper of good quality should contain at least 95% α - cellulose and should have only a very slight adsorption capacity. 5

6. PROCEDURE 1)A long strip of filter paper is moistened with a suitable buffer solution of the desired pH and the sample is applied transversely across the central part of the strip. 2)Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes. 3)Electric field of about 20 volts/cm is established. 4)The charged particles of sample migrate along the strip towards respective electrodes of opposite polarity , according to net charges, sizes and interactions with the solid matrix. 6

7. 5) Homogeneous group of particles migrate as a separate band. 6) The electrophoresis is carried out for 16-18 hours. 7 ) Separated Proteins are fixed to a solid support using a fixative such as Acetone or Methanol. 8) Proteins are stained (bromophenol blue) to make them visible. 9) The separated proteins appear as distinct bands. 10) Drawback-long time interval and blurring of margins. 7

8. OBSERVATION 1)The different fractions appear as blue coloured bands across the filter paper starting from the moving boundary backwards. 2)If a quantitative estimation is required for each fraction, the bands may be carefully cut and eluted , or the bands may be scanned optically in a densitometer . 3)In human plasma five different bands can be identified on paper electrophoresis. 8

9. APPLICATION Serum analysis for diagnostic purpose is routinely carried about by paper elelectrophoresis. Muscle proteins , egg white proteins , milk proteins & snake , insect venom analysis done by this technique. 9

10. THANK YOU…. 10