The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence

Antibodies

All Profilin-1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly.¹⁷ Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Peroxidase-conjugated monoclonal anti-Flag M2 is from MilliporeSigma (cat. number A8592). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429). The anti-FLAG (M2 clone) conjugated with Cy3 is from MilliporeSigma (cat. number A9594).

Cell culture

Both HAP1 WT and PFN1 KO cell lines used are listed in Table 1, together with their corresponding Research Resource Identifiers (RRID), to ensure the cell lines are cited properly.¹⁸ 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).

Antibody screening by Western blot

Western blots were performed as described in our standard operating procedure.¹⁹ HAP1 WT and PFN1 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) (Thermo Fisher Scientific, cat number 0089901) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429). 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 (GeneDireX, cat. number PM008-0500) was used.

Western blots were performed with precast midi 10% Bis-Tris polyacrylamide gels (Thermo Fisher Scientific, cat. number WG1201BOX) ran with MES SDS buffer (Thermo Fisher Scientific, cat. number NP000202), loaded in LDS sample buffer (Thermo Fisher Scientific, cat. number NP0008) with 1× sample reducing agent (Thermo Fisher Scientific, cat. number NP0009) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show them together with individual Western blots. 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 Signalling, 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) 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.²⁰ 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 A- (for rabbit antibodies) or protein G- (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively) or anti-Flag M2 magnetic beads from MilliporeSigma (cat. number M8823). Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.

HAP1 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) from Thermo Fisher Scientific (cat. number 87788), supplemented with protease inhibitor from MilliporeSigma (cat. number P8340). 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. Following centrifugation, 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 10% Bis-Tris polyacrylamide gels as described above.

Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure.^12–14 HAP1 WT and TMEM106B KO were labelled with CellTracker^TM green (Thermo Fisher Scientific, cat. number C2925) or CellTracker^TM deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated in 96 well glass plates (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 140770-10ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed three 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% BSA, 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 Profilin-1 antibodies overnight at 4°C. Cells were then washed three times for 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 three times for 10 min with IF buffer and once with PBS.

Images were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a 20× NA 0.95 water immersion objective and scientific CMOS cameras (16-bit, 1.97mm 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, 692/40 nm) to excite DAPI, CellTracker^TM Green, Alexa fluor 555 and CellTracker^TM 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 (MetaXpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTracker^TM channels using CellPose 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.²¹ Masks were used to generate cell outlines for intensity quantification. Background was subtracted using a minimum projection of all images collected in the Alexa fluor 555 channel. Images shown for the Alexa fluor 555 channel were modified by applying a Gaussian blur filter with a radius value of one pixel. Figures were first assembled with Adobe Photoshop (version 24.2.1) to adjust contrast then finally assembled with Adobe Illustrator (version 27.3.1).