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To enable elution of protein with little antibody contamination (for cleaner protein preparation and cleaner western blots), it is recommended to cross-link the antibody to the beads. An example procedure for this is shown below. The target protein should then be eluted with a mild eluent, such as glycine buffer.
Cross linking reagent:
Dimethyl pimelimidate (DMP)
Stock concentration 13 mg/mL DMP.
Working solution should be between pH 8 and pH 9.
1 M glycine (Add conc. HCl to correct pH to 3)
1mg/mL BSA in PBS
0.2 M triethanolamine in PBS (3.04 ml triethanolamine per 100 ml buffer)
50 mM ethanolamine in PBS (311.7 μL per 100 mL)
The antibody-bound beads can now be used in a normal IP procedure.
Elution of bound antigens:
To prevent elution of antibody with the target protein, use a gentle glycine elution gradient (up to 1 M).