3.2 - Dialysis

Before being able to analyze the structure of a protein, we must be able to purify a sample of proteins. Dialysis is a method of purifying a protein sample by removing small particles like salts, solvents from a protein containing solution. It’s essentially a prep step before other techniques in protein structural analysis.

Dialysis

• Purpose: Separate small molecules from larger protein molecules using a semi-permeable membrane.
• Example: Removing salts from protein solutions.
• Process:
  • Protein solution in a dialysis bag diffuses through the membrane.
  • Small molecules exit, proteins are retained.

To practice in an exam setting, one of our workshops (actually a few questions were on this) was on calculating dialysis. The example used in the lecture was:

The dialysis bag (membrane) has a MW cut-off of 1000, and contains a 4 mL solution of 20 mM Tris. HCl at pH 7.5, 0.6 M NaCl, 10 mg/mL protein (20 kDa). The bulk solution is 1000 mL of 20 mM Tris.HCl at pH 7.5. What is the NaCL inside the bag after dialysis

So we know the proteins will not be able to pass through the membrane, the proteins have a 20kDa and the membrane has a MW cutoff of 1000. We also know that $NaCl$ is lighter than the MW so it can pass through the membrane freely.

The equation for the *conservation of moles =

$$Molesbeforedialysis=Molesafterdialysis$$ $$C_i\times V_i=C_f\times (V_i+V_o)$$

Now to find for $C_f$

$$C_f=\frac{C_i\times V_i}{V_i+V_o}$$

So to find the $NaCl$ in the bag, you need to calculate

$$C=\frac{0.6M\times 4mL}{1000mL+4mL}$$ $$C=0.00239M$$

Topics related: Protein Science Map 3.1 - Protein diversity & techniques to study proteins 3.3 - Techniques and Reagents 3.4 N-Terminal amino Acid Sequencing