For extracting Lysozyme from egg white which method would you recommend and please help me understand why.
Lysozyme has a higher isoelectric point (pH 10.5) and a lower molecular mass (14 307 Da) than most proteins so both these methods make quite a bit sense. Use Sephahdax G100 for gel permeation chromatography and CM-Sephadex G25 ion exchanger for ion exchange.
From what I can understand, Gel Permeation will separate the egg white based on Mr of the various proteins and compounds. There is quite a bit of variation in the Mr of egg white proteins but lysozyme, in fact, doesn't have the lowest Mr as Cystatin and Ovomucoid have lower Mr. Furthermore, GPC requires around at least a 10% difference in molecular weight for a reasonable resolution of peaks to occur. The range of Mr is also quite broad in this method. Will this be disadvantageous perhaps? So GPC does have the plus side of it having a well-defined separation time due to the fact that there is a final elution volume for all unretained analytes.
Now Ion exchange chromatography is very specific in the nature of how the exchanger interacts with the mixture being separated. Lysozyme has a PI of 10.7 which is much higher than the rest of the proteins in the egg white. As with GPC we use 0.05M Tris-EDTA, pH 8.2, containing 0.05M NaCl as the standard. Can this setup result in Gibbs Donan effect or chromato- focusing ?
I'm currently leaning towards GPC as the option to yield the purist product especially seeing as it can be desalted afterwards with G25.
Please help guide me in the right direction. I'm quite new to this but am ready and very willing and keen to learn and expand my abilites.