Identification of high-performing antibodies for Apolipoprotein E for use in Western Blot and immunoprecipitation

Antibodies

All Apolipoprotein E antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly.¹⁴ All antibodies tested detect Human ApoE. Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from Thermo Fisher Scientific (cat. number 65-6520 and 62-6120).

Cell culture

HAP1 WT and APOE KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.¹⁵ Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 μg/mL streptomycin (Wisent cat. number 450201). Cells were starved in DMEM high-glucose containing L-glutamate and penicillin/streptomycin.

Antibody screening by Western Blot on culture media

HAP1 cells WT and APOE KO were washed 3× with PBS 1× and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500× g to eliminate cells and larger contaminants, then for 10 min at 4500× g to eliminate smaller contaminants. Culture media were concentrated by centrifuging at 4000× g for 30 min using Amicon Ultra-15 Centrifugal Filter Units with a membrane NMWL of 10 kDa (MilliporeSigma cat. number UFC901024).

Western Blots were performed as described in our standard operating procedure.¹⁶ Midi precast 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) were used and proteins were transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0.1% Tween 20 (TBST) (Cell Signaling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) or with Clarity Western ECL Substrate from Bio-Rad (cat. number 1705061) prior to detection with the iBright™ CL1500 Imaging System from Thermo Fisher Scientific (cat. number A44240).

Antibody screening by immunoprecipitation on culture media

Immunoprecipitation was performed as described in our standard operating procedure.¹⁷ Antibody-bead conjugates were prepared by adding 2 μg or 20 μL of antibody at an unknown concentration to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein G - (for Mouse and rat antibodies) and protein A - (for rabbit antibodies) from Thermo Fisher Scientific (cat. number 10003D and 10002D, respectively). Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) was supplemented with the Halt Protease Inhibitor Cocktail 100X from Thermo Fisher Scientific (cat. number 78446) at a final concentration of 1×. Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies. Starved HAP1 WT culture media were concentrated as described above. 0.6 mL aliquots at 1.5 mg/L of protein were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP Lysis Buffer and processed for SDS-PAGE and Western Blot on precast midi 4-20% Tris-Glycine polyacrylamide gels. Prot-A: HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a concentration 0.4 μg/mL.