Figure 3.

Dual streptavidin- and calmodulin-based purification. A: First step streptavidin-based purification recovers substantial Btk protein: supernatant of whole cell lysate (WCL); unbound portion (SBP-Unb.); and product of 2 mM biotin eluate (Bio-Elu.). B: Protein recovery as illustrated by serial dilution of biotin eluate: undiluted (Bio-Elu.); twofold, fourfold, sixfold, and eightfold dilution; and supernatant of whole cell lysate (WCL). C: Second phase of TAP purification via calmodulin binding is ineffective to recover Btk: biotin eluate resulted from the first round of purification (Bio-Elu.); unbound portion following incubation of streptavidin eluate with calmodulin resin (CBP-Unb.); product of 5 mM EGTA elution (EGTA-Elu.). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]