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Summary: 101
DEAE Chromatography of Antibodies
· Plan to start with 5 mls of acites fluid, or an ammonium sulfate cut from 5-10 mls of ascites
fluid. If starting with ascites fluid, it must be extensively dialyzed against 10 mM Tris, pH 8.5.
Use SpectraPor 2 (12-14 kDal cut off) tubing. Use twice as much dialysis tubing as needed for the
dialyzed volume. This will allow enough space for expansion during dialysis. Use tubing clips at the
ends. Dialyze overnight at 4°C in at least 100 volumes of buffer. Change buffer three times.
1. Pour a 15 ml column of DEAE matrix (DEAE Sepharose or DE52). Use 10 mM Tris, pH 8.5. Do
this in the cold room. Set up as in Figure below.
· Alternatively, regenerate a previously used DEAE column. See Harlow and Lane for procedure.
· Tris-HCl at pH 8.5 at 25°C will have a pH of ~9 at 4°C.
· Use a flow rate = ~1 ml/minute for the steps below.
Stirplate Pump Fraction Collector
Column
25250
Gradient
Former
2. Wash column with 10 volumes of 10 mM Tris, pH 8.5.
3. Bind antibody to column. Save load eluate.
4. Wash with 10 volumes of 25 mM NaCl in 10 mM Tris, pH 8.5.
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