All synthetic membranes have holes: balloons have them, saran wrap has them, eve
ID: 152069 • Letter: A
Question
All synthetic membranes have holes: balloons have them, saran wrap has them, even condoms have them. Don't worry about these holes because they are actually tiny pores too small to allow the passage of sperm or viruses in the last example. The process of stretching membranes to make them thinner and their chemical composition introduces these pores. Scientists use this to their advantage. The membranes you used in general biology labs (called dialysis tubing) had a pore size that allowed molecules that were 13 kDa in size or smaller to diffuse freely across the membrane. This pore size is referred to as the molecular weight cut off, or MWCO. Given that our protein of interest, lysozyme, is 14 kDa, propose mechanisms that would use these dialysis tubing and the concepts of osmosis and diffusion to solve the following problems Assume you have dialysis tubing of any MWCO, and the ability to make solutions of any molarity 1. The protein you purifiled is now stored in the high salt buffer (0.75 M NaCI). You are all well aware that salt can denature proteins. Propose a set up that would allow you to reduce the salt concentration. Now that you have reduced the salt concentration, you have another problem. SDS- PAGE shows a contaminating protein, ovalbumin. Total protein in the egg white comprises 54% of this 45 kDa protein. Propose a set up that would reduce the concentration of this contaminating protein. 2. 3. Now that you have reduced the contaminants, you have a very dilute lysozyme solution. Propose a mechanism that would increase the concentration of lysozyme. (Hint: it does not involve lysozyme at all!)Explanation / Answer
1: Putting the dialysis tubing filled with high salt buffer and desired protein in a low salt buffer (less than 0.75 M NaCl) will direct the movement of water into the dialysis tubing which in turn would lead to reduce the concentration of salt in the buffer and prevent the denaturation of protein. Movement of water from the region of its higher concentration to that of lower concentration is called as osmosis.
2: The membrane is stretched to increase the pore size that would allow the diffusion of 45kDa protein. The dialysis tubing is put in the solution containing lysozyme but no ovalbumin. Diffusion of ovalbumin through the pores of membrane would reduce the concentration of this protein in the desired protein.
3: The membrane of the dialysis tubing is brought to the normal pore size and is put in the higher salt buffer to allow the exosmosis of water from dilute lysizyme solution (filled in dialysis tubing) to the higher salt buffer. This would lead to increase the concentration of lysozyme as solvent has moved through osmosis into the surrounding solution.