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Lab Activity 1

Lab Activity 1. Dialysis of Proteins IUG, 2012 TMZ. Dialysis. Passage of solutes through a semi-permeable membrane. Pores in the dialysis membrane are of a certain size.

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Lab Activity 1

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  1. Lab Activity 1 Dialysis of Proteins IUG, 2012 TMZ

  2. Dialysis • Passage of solutes through a semi-permeable membrane. • Pores in the dialysis membrane are of a certain size. • Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

  3. When is dialysis recommended? • If working with proteins and nucleic acids (elimination of small molecular weight substances such as reducing agents such as DTT, BME, labeling reagents or preservatives (e.g. sodium azide). 2. Exchanging buffers for electrophoresis, ion exchange or affinity chromatography.

  4. Dialysis • Following a salting-out step, the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps. • The salt can be removed by dialysis – dialysis tubing has pores with a specific molecular weight cut-off that allows smaller molecules (salt) to pass. Buffer– large volume Dialysis tubingwith protein and high salt Exchange buffer > 3 times

  5. Experiment • Reagents • Saturated solution of Ammonium Sulfate • 5% aqueous solution of barium chloride • Biuret reagent • Egg white ( an aqueous solution) or blood serum sample

  6. Materials • Cellophane cut into sheets of 12.5 x 12.5 cm • A beaker with distilled water • Glass rods • Small rubber rings • A test tube stand with a set of test tubes pipettes

  7. Procedure.. • To 5 ml of egg white soln ( or blood serum) in a test tube, add a couple drops of sat., (NH4)2SO4, then stir with a glass rod. • Sample 10 drops of the mixture into 2 test tubes; use them to perform the biuret test and the SO42- test. • To perform the test for sulfate ion, add 2 to 3 drops of barium chloride. • Shape the cellophane sheet into a sack, then fill it up to 1/3 of its volume with the protein solution.

  8. Procedure.. 5. Clip the sack edges between 2 glass rods tightly held against each other with rubber rings that clasp the glass rods at their ends. 6. Place the cellophane sack into distilled water, so as to keep the sack submerged in water. The level of protein solution in sack must be below the water level in the beaker. 7. Let the dialysis proceeds for about 1 h. then take 2 samples (10 drops each) of the liquid contained in the beaker (dialyzer) to perform the biuret test for protein & sulfate ion.

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