Lab #3: Ion Exchange Chromatography

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Lab #3: Ion Exchange Chromatography
The purpose of this experiment was to separate proteins on the basis of their net charge at a particular pH. In cation exchange chromatography positively charged molecules are attracted to a negatively charged column. Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged column. Experimental results could be monitored in a predictable way by controlling running pH, salt concentration, and by selecting the type of ion exchanger.
Procedure: all procedures are listed in the lab manual.
Table 1: Abs 280 Raw Data
Dilution Factor Measured Abs280 Undiluted Abs 280
(B x C) Graph Bar
HEW 80 0.918 73.44
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5. Present an estimate of lysozyme’s net charge at pH 11. The carb 1 fraction which is mainly constituted of lysozyme is near pH 11 and the net charge of lysozyme is slightly negative.
6. The 200 mM (0.2 M) Na2 CO3 buffer contains much higher [ ] of Na+ than the 50 mM sodium phosphate buffer. When changing from the phosphate buffer to the carbonate buffer, the difference in Na+ will hinder the desired effect since positive Na+ will compete for binding sited on the negative column.
7. Student’s attempt to make lysozyme bind tighter to CM Sephadex by starting pH lower than 7 is unsuccessful because the charged groups on the column itself are also titratable groups with specific pKa values. At pH 3 the COO – group of CM column will become protonated and will no longer are able to bind proteins.

Ion exchange columns (beads) work by having a fixed charge on their surface which, before protein added, is neutralized by soluble counterions (like chloride or sodium in our case) from buffers. As previously learned, most proteins contain charged amino acids on their surfaces. Even if proteins have an equal number of positively-charged and negatively-charged amino acids on the surface, they 're never exactly evenly distributed which means that there are areas on the surface of the protein which have an overall positive or negative charge to them.

In our experiment, positively-charged patches on the
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