Sepharose 4b column

Three antibodies against cMLCK were generated. Two polyclonal antibodies against mouse cMLCK were raised in rabbits using recombinant-GST-cMLCK(41–448aa) [cMLCK(41–448)] and recombinant-GST-cMLCK(747–795aa) [cMLCK(747–795)] as antigens. These antibodies were preabsorbed by the GST affinity column, then affinity-purified using a CNBr-activated Sepharose 4B column (GE) conjugated to the recombinant. Hybrid peptide (42-55/93-102aa, namely, WPEVLELVRA-aminohexanoic acid-LLHFQEDVTEKLQC) [cMLCK(42-55/93-102)] was synthesized and coupled with KLH. Theses amino acid sequences in cMLCK were conserved among human, bovine, dog and rodents. This rabbit anti-cMLCK antibody was prepared and affinity-purified using Epoxy-activated Sepharose 6B column (GE).

An EasySep™ human basophil enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) was used to negatively purify human basophils to obtain a purity of greater than 90% using flow cytometry (30 (link)).
For activation analysis, human basophils isolated from healthy controls were aliquoted at 4,000 cells/well in a total volume of 200 µl medium containing IL-3 (2 ng/ml, PeproTech., London, UK); to these were added the AB serum of healthy controls (20% of a total volume) or newly diagnosed patients with SLE (20% of a total volume), or CICs that were precipitated by PEG 6000 (3.75%) from the serum of newly diagnosed patients with SLE (31 (link)), or serum from newly diagnosed SLE patients that flow through a Sepharose 4B™ column (GE Healthcare, Uppsala, Sweden) coupled with anti-IgE antibody to depletion of IgE (32 (link)), or anti-IgE antibody (0.5 µg/ml; positive control) (Abcam Inc., Cambridge, MA, USA) at 37°C for 15 min (CD203c detection) or 24 h (CD62L, CCR7, and IL-4-positive detection) (33 (link)).