Subsequently, by further washing with TBS-T, protein bands were visualised with an enhanced chemiluminescence system using a Bio-Rad Chemidoc Apparatus.
Pre-diluted peroxidase-polymer labelled goat anti-mouse/rabbit secondary antibody (Envision™, Dako) was applied for 30 min at room temperature, followed by further washing with buffer to remove unbound antibody.
Prediluted peroxidase polymer-labelled goat anti-mouse/rabbit secondary antibody (Envision™, Dako) was applied for 30 min at room temperature, followed by further washing with buffer to remove unbound antibody.
The beads were then washed three times with 20 mM Tris, pH 8.0, 200 mM KCl, 10% glycerol, followed by three further washes with 50 mM Tris, pH 7.5, 150 mM potassium acetate, and 2 mM MgCl2, and a final wash in 50 mM Tris, pH 7.5.
Incubation of the resin with the cellular lysate for 1 h at 4°C was followed by two washes with mammalian lysis buffer A, and two further washes with 1× PBS to give a pure M2-Flag-WNK4 slurry.
After two further washes with PBS-T, immunoreactive proteins were identified by enhanced chemiluminescence.
After further washes with blocking buffer and PBS, immunoreactive proteins were visualised by enhanced chemiluminescence.
Unbound particles were removed with further washes with PBS-T.
Following overnight incubation, the chitin/resin slurry was returned to the column and the liberated semisynthetic enzyme was eluted via gravity flow by further washing of the resin with chitin buffer B. The semisynthetic enzyme was concentrated and buffer exchanged with buffer C [10 mM Tris-EDTA, 10 mM NaCl, and 20 mM βME (pH 8.0)] via ultrafiltration (Amicon Ultracel 30 kDa cutoff filters).
