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SUPPLEMENTAL MATERIAL & METHODS
Conjugation of antibodies to biotin. Commercial available antibody preparations free from carrier
proteins (such as gelatin, BSA, casien, or serum) or stabilizers (such as Tris, glycine, lysine, ethanolamine,
sodium azide, glycerol, etc.) were conjugated to biotin using a Sulfo-NHS-LC-Biotin kit (Thermo
Sientific, Pierce EZ-Link) per manufacturer recommended procedures using a 25:1 biotin:antibody ratio.
Excess biotin was removed by 10 kDa molecular weight cut-off dialysis membrane (Thermo Scientific) in
PBS for 6 hours at 4ºC. Biotinylated antibody solutions were supplemented (2.5% by volume) with PBS
containing 4% BSA and 2% Proclin 300 (Supelco) as a preservative and stored at 4ºC.
Linked heterodimer fusion proteins
Linked heterodimer fusion protein constructs for Bcl-xL–Bak, Mcl1–Bak, and Bax–Bcl-2 were made using
synthetic DNA (Life Technologies) with Gateway attB1 and attB2 sites flanking the insert region. His6MBP (maltose binding protein) tags were used to enhance solubility and permit affinity purification of the
final dimers. The Entry clones were subcloned into pDest-636 Gateway LR recombination (Life
Technologies) to create the final heterodimer baculovirus expression construct. All clones were sequence
verified throughout the entire cloned region. The expression clones were transformed into E. coli
DH10Bac (Life Technologies), and plated on selective media containing gentamycin, kanamycin,
tetracycline, IPTG, and X-gal per the manufacturer’s protocols. White colonies were selected from these
plates, and bacmid DNA was generated by alkaline lysis plasmid preparation and verified by PCR
amplification across the bacmid junctions. Proteins were expressed in either insect SF5 or HEK293
mammalian cells using the baculovirus expression vector system. TEV protease was used to cleave off the
His6-MBP tag, and a second round of affinity purification and ion-exchange chromatography were used to
obtain the final proteins.
Immunoprecipitation and Western blotting for determination of antibody specificity
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Cell lysate samples (500 g) were incubated overnight at 4ºC with capture antibody linked to Luminex
magnetic beads or Protein A/G agarose. Beads were washed with PBS, eluted with SDS-PAGE buffer
(Life Technologies), and run on SDS-PAGE (BioRad). Gels were transferred to nitrocellulose membranes
and transferred membranes were blocked with 4% BSA and incubated overnight with the detection
antibody. After washing the membrane with PBS, the blots were probed with anti-species specific HRP
conjugated secondary antibodies (Jackson Immuno). Signal was generated with ECL substrate (Protein
Simple) and images were captured using the FluorChemE instrument (Protein Simple).
THP-1 (leukemia), PC3 (prostate cancer), LS-141 (liposarcoma), and HCT 116 (colorectal carcinoma) cell
lines were used with in vitro drug treatments to demonstrate the specificity of capture and detection
antibodies by immunoprecipitation/western blotting (IP/WB) experiments (Supplemental Fig. S1).
Experimental drugs ABT-263, ABT-199, and gossypol were purchased from commercial providers (R&D
Systems).
Evaluation of pre-analytical factors. To test stability, six different aliquoted lysates were frozen
at -80°C as a control, and kept at 2-8°C for 2, 4, or 24 hours or 20-25°C for 4 or 24 hours. Samples were
analyzed without further freeze/thaw cycles (Supplemental Fig. S2). To test freeze/thaw effects, aliquots
were frozen at -80°C and then quickly warmed to 20-25°C once, twice, or three times. For both
evaluations, six aliquots were used for each time point and analyzed using all three multiplex
immunoassay panels (Supplemental Fig. S3).
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