You have a 10mL preparation of a bioproduct and wish to perform dialysis to desa

Question

You have a 10mL preparation of a bioproduct and wish to perform dialysis to desalt (remove NaCl) while keeping a 100 kDa protein However, the buffer solution which you use to perform the dialysis is expensive, as is personnel tune for each change of solution Design a protocol to achieve a 1000x dilution of NaCl, minimizing the overall "cost". Identify equipment and supplies, make a schematic of the situation, define variables and parameters, perform an analysis of transport kinetics, assume values as needed, and provide reasoning for your procedure.

Explanation / Answer

Dialysis

Introduction: Proteins are small molecules remove or changing the buffer in a dialysis membrane. It is easy and inexpensive method. Dialysis tubing is a semi permeable membrane that can be purchased with a specified range of pore sizes. Dialysis of proteins purification occurs up to at 10, 000 Daltons. Dialysis mechanism was used to remove impurities from purification sample. Most dialysis tubing has a molecular weight cut-off of 5,000 to 10,000 Daltons.

Principle: Dialysis tubing is a semi-permeable membrane available in a wide range of size dimensions and pore sizes (molecular weight cut-offs). Our procedure uses dialysis tubing with a 10,000 Dalton molecular weight cut-off. Our extract is placed inside the tubing and the ends are sealed off. The tubing is then suspended in a large volume of the desired buffer solution. The pores in the membrane allow molecules that are smaller than the pores to move freely across the membrane. Therefore, the ammonium sulfate ions will cross out of the tubing into the buffer. Eventually, an equilibrium is achieved where the concentration of ammonium sulfate is equal inside and outside of the tubing. However since the volume outside the tubing is much greater than inside, and this outside volume is replaced with fresh buffer, over time, most of the salt will leave the tubing. Larger molecules (such as most proteins) are retained within the membrane. Since most buffer components are small molecules and can therefore pass through the pores, dialysis is also used as a method for changing buffers.

Materials:

1 L Bottles

Large bottle Centrifuge & Normal

LB

50 mL Conical Tubes

French Press Cell and Machine

Plastic Transfer Pipets

Ice Bucket with Ice

White Oak Ridge Tubes

4L Beaker

Dialysis Tubing and Clamps

10 mL syringe

Needle to Insert Sample

Spectrophotometer

Cuvettes

Refrigerator

Dialysis membrane

Buffer: 0.1 M sodium phosphate mono basic and 0.1 M sodium phosphate di basic

Procedure:

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