Pack the column with 4 ml of IgG Sepharose 6 FF suspension to obtain a 2 ml column.
Wash the column with 5 bed volumes of Tx-Swb prior to use in order to remove traces
of EtOH.
Equilibrate the column with 2-3 bed volumes each of:
0.5M HAc, pH 3.4
Tx-Swb
0.5M HAc, pH 3.4
Tx-Swb
Check the pH of the eluate with pH paper and adjust pH of sample is necessary (both should be neutral). Apply the sample to the column.
Wash the gel with:
2 bed volumes of Tx-Swb
5 bed volumes of Tris-NaCl (0.1M) *
2 bed volumes of 5 mM NH4Ac, pH 3.4
5. Elute the
sample with 0.5M HAc, pH 3.4 6. Collect the
first 15ml as eluate 7. Re-equilibrate
lgG Sepharose 6 FF with 5-10 bed volumes of Tx-Swb
until effluent is above pH 7.0.Note: Wash off Triton,
because it absorbs in the UV range. When reusing gel after this point, it
requires washing with 50ml Tx-Swb before washing with Tris-NaCl beginning at
step #4.
Note: Wash off Triton, because it absorbs in the UV range. When reusing gel after this point, it requires washing with 50ml Tx-Swb before
washing with Tris-NaCl beginning at step #4.
