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 an equal 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 withdraw supernatant 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 .
