For monoclonals use Protein G column
For polyclonals use Protein A column
1. Using a vacuum flask, wash the sepharose beads in three to five volumes of cold PBS. Avoid air getting into the slurry.
2. Pour beads into a 15ml column (Biorad), set up in cold room.
3. Thaw antisera on ice to avoid precipitation – if you see precipitates forming in the tube, centrifuge at 15,000g for 5 minutes. Take supe, avoiding lipid layer. Alternatively, collect hybridoma supernatants by centrifuging at 15,000g for 15 minutes. Avoid cell pellet.
4. Apply sera to column. Avoid letting column run dry. Aim to load the column to 90% capacity, which is approx 18ml of 5mg/ml (typical serum concentration) on a 5ml column. Keep flow through for later analysis – not all of the IgG in the serum may bind to the column.
5. Pour flow through onto column for a second time to maximize binding.
6. Wash 3 times in cold 100mM Tris pH 8.0
7. Wash 3 times in cold 10mM Tris pH 8.0
8. To elute bound protein, add 1ml of 100mM glycine pH 2.9 to the column. Collect flow through in a 1ml tube – this is your first fraction. Collection tubes should contain 0.5ml of 1M Tris pH 8.0, to neutralize the eluate immediately after elution from the column. Repeat until you have at least 5 fractions.
9. Assay the protein content of each fraction using Bradford protein assay. Use IgG as a standard, not BSA (from Sigma).
10. Pool fractions containing protein and store in aliquots at 1mg/ml (if you have that much) at –20oC. You may add sodium azide (to prevent bacterial growth) and/or BSA (0.1mg/ml, this is especially important for IgG solutions below 0.1mg/ml) at this point. Do not freeze/thaw your purified antibody or store it above freezing, as this will encourage degradation.
11. Regenerate sepharose by washing extensively in PBS, followed by overnight incubation in 2% Ethanol. Wash in PBS before use.
For phospho-specific antibodies, you may purify the phospho-specific IgG using a phospho-peptide column. You may also try subtracting IgG which recognizes the non-phospho protein, by using the non-phosphorylated peptide and collecting the flow through.
1. Weigh 5-10mg of the antigenic peptide and dissolve in 0.5ml DMSO.
2. Collect equal volumes of Affygel 10 and 15 (Biorad, numbers refer to the length of the crosslinker arm on the Affygel beads). Wash in 5 volumes of DMSO in a vacuum flask. Avoid getting air into the slurry.
3. Combine peptide and Affygel and incubate at room temp overnight with rocking.
4. Wash beads three times in 100mM Tris pH8.0.
5. Add 100mM Ethanolamine to beads (blocking step). Incubate at room temp overnight with rocking.
6. Wash beads three times in 100mM Tris pH8.0.
7. Thaw antisera on ice as above or collect hybridoma supernatants by centrifuging at 15,000g for 15 minutes. Avoid cell pellet.
8. Combine antisera and beads. Rotate overnight at 4oC.
9. Following day, pour slurry into a 15ml column (Biorad) set up in cold room. Avoid getting air into column. Collect flow though in case it contains non-bound antibody.
10. Repeat steps 6 to 11 above.