Immunopurification — Small Scale using Epitope Tag Affinity Matrices
Combine affinity matrix and IP Buffer in a microfuge tube at a 1:5 dilution.
Add tagged protein to mixture.
Gently rock aliquots for one hour at 4°C.
Centrifuge mixture at 10,000g for 20 seconds at 4°C.
Remove supernatant without disturbing beads.
Add anequal volume of IP Wash Buffer to the beads and re-suspend matrix.
Gently rock aliquots for 20 min.
at 4°C.Keep a portion of the supernatant from each rinse step to use in Western blot analysis.
Repeat step 2 four times.
Elute the bound protein with the appropriate epitope peptide at 1 mg/ml concentration in 50 mm Tris HCl(pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8.0).
Resuspend beads, incubate, centrifuge and withdrawsupernatant as in step 2, repeating for a total of four elutions.
Recover as much of the eluate as possibleat each stage.
For each elution sample, prepare at 1:1 dilution with reducing gel loading buffer.
Boil tubes for 3 min.
Analyze the supernatant samples by Western blot.
