A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence

Antibodies

All PLC-gamma-2 antibodies are listed in Table 2, along with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.³² Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).

Cell culture

Both THP-1 and PLCG2 KO cell lines used are listed in Table 1, together with their corresponding RRID to ensure the cell lines are cited properly.³³ Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, cat. number 11875119) 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).

One set of the THP-1 WT and PLCG2 KO cells were treated with 200 ng/mL of PMA (Abcam, cat. number ab147465) for 2 days. 200 ng/mL of PMA was added to fresh medium on both day 1 and day 2.³⁴ The other set of cells, WT and PLCG2 KO remained untreated.

Antibody screening by Western Blot

Western Blots were performed as described in our standard operating procedure.^27,28 THP-1 WT and PLCG2 KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.

Western Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and 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% milk in TBS with 0.1% Tween 20 (TBST) (Cell Signalling 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 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

Immunoprecipitation was performed as described in our standard operating procedure.^27,28 Antibody-bead conjugates were prepared by adding 10 μL of antibodies 3872 and 55512** or 2 μg of the remaining antibodies tested 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 A - (for rabbit antibodies) or protein G - (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). The low concentrations of antibodies 3872 and 55512** can account for why 10 μL was added to make the antibody-bead conjugates, rather than 2 μg. Tubes were rocked ~1 hr at 4°C followed by several washes to remove unbound antibodies.

THP-1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate 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 a precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific.

Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure.^27,28 THP-1 WT and PLCG2 KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated in 96-well plate with optically clear flat bottom (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37^oC, 5% CO₂. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10 ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095), 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary PLC-gamma-2 antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.

Images were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20× NA 0.95 water objective lens and scientific CMOS camera (16-bit, 1.97 mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose³⁵ v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns. Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).