SUPPLEMENTAL Material & Methods
Conjugation of antibodies to biotin. Commercial available antibody preparationsfree 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 aSulfo-NHS-LC-Biotinkit (ThermoSientific, Pierce EZ-Link)per manufacturer recommended procedures using a 25:1 biotin:antibody ratio. Excess biotin was removed by 10kDa 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 andstored at 4ºC.
Linked heterodimer fusion proteins
Linked heterodimer fusion protein constructs for Bcl-xL–Bak, Mcl1–Bak, and Bax–Bcl-2were made using synthetic DNA (Life Technologies) with Gateway attB1 and attB2 sites flanking the insert region. His6-MBP (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 GatewayLR 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
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 andtransferred membranes were blocked with 4%BSA and incubated overnight with the detection antibody. After washing themembrane 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 at80°C as a control, and kept at 2-8°C for 2, 4, or 24 hours or 2025°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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