Materials
WedgeWell Tris-Glycine 4-12% Mini Gels, SeeBlue Plus2 pre-stained
molecular weight standards and 1 M Tris-HCl (pH 8) were purchased from
Invitrogen of Thermo Fisher Scientific (Waltham, MA). Dynabeads MyOne
Streptavidin T1 and Dynabeads Antibody Coupling Kit was purchased from
Invitrogen of Thermo Fisher Scientific (Waltham, MA). Trans-Blot Turbo
Transfer Pack was purchased from Bio-Rad (Hercules, CA). Formic acid,
acetonitrile, Diothiothreitol (DTT) and 1-step ultra TMD-blotting
solution were purchased from Thermo Fisher Scientific (Waltham, MA).
Acetic acid, 10X Tris buffered saline (TBS), Iodoacetamide (IAM), bovine
serum albumin (BSA) and urea were purchased from Sigma-Aldrich (St.
Louis, MO). HEPES buffered saline with EDTA and 0.005% v/v Surfactant
P-20 (HBS-EP) was purchased from GE (Boston, MA). All monoclonal
antibodies, recombinant CHO PLBD2, anti-PLBD2 monoclonal antibody were
prepared in house at Regeneron Pharmaceutical. Inc. Polysorbate 20 and
polysorbate 80 were purchased from Croda and aliquoted in Regeneron
Pharmaceutical. Inc. Human PLBD2 was purchased from Origene Technologies
Inc (Rockville, MD). Sequencing Grade Modified Trypsin was purchased
from Promega (Madison, WI). Anti-goat IgG antibody was purchased from
Abcam (Cambridge, UK). Oasis Max column, Acquity UPLC BEH C4 column,
Acquity UPLC CSH C18 column were purchase from Waters (Milford, MA).
Acclaim PepMap 100 column and Acclaim PepMap RSLC column were purchased
from Thermo Fisher Scientific (Waltham, MA). DPBS (10x) was purchased
from Gibco by life technologies and Tween20 was purchased from J.T.
Baker (Phillipsburg, NJ). Q-Exactive Plus with electrospray ionization
(ESI) source was purchased from Thermo Fisher Scientific (Waltham, MA).
Antibody mAb-1 is an IgG4 antibody expressed from a CHO K1 cell line;
mAb-2 is the same IgG4 antibody as mAb-1 but expressed from a CHO cell
line harboring a PLBD2 gene knocked out; mAb-3 is mAb-1 processed, as
described below, to remove PLBD2; mAb-4, mAb-5, mAb-6 and mAb-7 are
different IgG4 antibodies without the PLBD2 removal purification step;
mAb-8 is an IgG1 antibody without the PLBD2 removal step.