Most of the information you’ll ever need is provided on the Amersham website (under Literature » Handbooks). It covers basic principles of protein purification, basic theory of chromatography, how to pack columns, assess packing efficiency, recommended flow rates etc. Other documents on these topics are:
One important consideration in gel filtration chromatography is to always use a minimum of 0.15 M NaCl in your elution buffer to reduce non-specific electrostatic interactions between the protein sample and the chromatography matrix.
The chromatography matrices we have are high grade and therefore very expensive. They are also fairly robust, but will not tolerate drying out. Our matrices are Sepharose and Sephacryl anionic exchangers. Recently we got sulfopropyl (SP) cationic exchanger.
Storage of matrices
- Either in 20% ethanol [Sephacryl and Sepharose] or
- 10 mM NaOH [Sepharose] or
- 20% ethanol, 0.2 M sodium acetate [sulfopropyl]
Cleaning of columns
- Add half a column volume of 0.5 M NaOH [in distilled water] and allow to contact the matrix for 1 hour. Chase this with 2 column volumes of buffer to neutralise the pH. The column is ready for use.
- For anion exchangers, NaOH alone may not be enough to remove DNA. You would need to use 0.5 M NaOH combined with 1 M NaCl. Very heavy DNA contamination may also require DNAse treatment.
- For cationic exchanger (SP), 2 M NaCl may be sufficient to remove all bound proteins on regular basis. For rigorous cleaning, 2 M NaCl, followed by 1 M NaOH and finally 20% ethanol are recommended. Water should be used in between.