IgG Purification Protocol
IgG Purification
Note: Content of gel is 1 ml of gel in each column
- Wash columns with 10 ml of 1 X PBS.
- Add 2.5 ml of serum to 2.5 ml of PBS and divide this mixture into two IgG columns.
- Collect the serum into a test tube as it drains through the column.
- When serum finishes draining, pour the washed serum back into the column and begin collecting flow through again. Repeat this step 5 to 6 times.
- Wash the columns with 10 ml of 1 X PBS.
- Prepare several 1 ml tubes with 50µl of 1M Tris (pH = 9.5).
- Add 1 ml of Elution Buffer (100mM Glycine pH=2.8) to each tube and collect 1 ml of flow through.
- Move to the next prepared tube and repeat step 7.
- Test each 1 ml sample by preparing ELISA plate with 10µl of Bradford Assay and add 50µl of each 1 ml flow through. Keep the samples that change the Bradford Assay from red to blue.
- Dialyze the positive 1 ml samples together in 4 Liters of 1 X PBS at pH=7.2 for at least 24 hours.
- Use spectrometer at 280 nm to find concentration of IgG in solution. (Extinction coefficient = 1.4)
- To store IgG solution, keep frozen at -4°C to -20°C.
Print
10/12/2007