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

Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.


Introduction
Profilin-1, encoded by the PFN1 gene, is ubiquitously expressed and the most abundant of the Profilin genes. 1 It plays an essential role in the polymerization of actin by binding and sequestering actin monomers, indicating its cytoskeletal function. 2,3 At high concentrations, Profilin-1 act as an inhibitor of actin polymerization whereas at low concentrations, it acts as a catalyst. 3,4 Profilin-1 is of recent interest thanks to the identification of PFN1 mutations in 25 human familial amyotrophic lateral sclerosis (fALS) patients. [5][6][7][8] PFN1 is among a group of ALS-related genes that directly affect cytoskeletal dynamics, namely TUBA4, ALS2, KIF5A and SPAST, hypothesizing that cytoskeletal dysfunction contributes to motor neuron degeneration. 9 PFN1 mutant mice that carry the G118V mutation have motor defects consistent with ALS pathology, suggesting that Profilin-1 molecular studies would provide insight into pathogenic mechanisms of motor neuron disease. 10 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.
Here, we compared the performance of a range of high-quality commercially available antibodies for Profilin-1 and characterized several antibodies for Western blot, immunoprecipitation and immunofluorescence experiments, enabling biochemical and cellular assessment of Profilin-1 properties and function.

Results and discussion
Our standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells. [11][12][13] The first step was to identify a cell line(s) that expressed sufficient levels of Profilin-1 to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we found to be a suitable cut-off (Cancer Dependency Map Portal, RRID: SCR_017655). Of all cell lines analyzed, commercially available HAP1 cells expressed the PFN1 RNA transcript above the adequate cut-off level. Parental and PFN1 KO HAP1 cells were obtained from Horizon Discovery (Table 1).
For Western blot experiments, we resolved proteins from WT and PFN1 KO cell extracts and probed them side-by-side with all antibodies in parallel 12,13 (Figure 1).
For immunoprecipitation experiments, we used the antibodies to immunopurify Profilin-1 from HAP1 WT cell extracts. The performance of each antibody was evaluated by detecting the Profilin-1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates 12,13 (Figure 2). For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. [12][13][14] In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3).
In conclusion, we have screened Profilin-1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies using our standardized experimental conditions. The underlying data can be found on Zenodo. 15,16 Methods 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).  An exception was given for antibody A9188** which was titrated to 1/30000, as the signal was too weak when following the supplier's recommendation. When the concentration was not indicated by the supplier, which was the case for AC-PFN1-4**, we tested the antibody at 1/1000. Antibody dilution used: 11680-1-AP at 1/6000, 67390-1-lg* at 1/30000, MAB7779* at 1/5000, NBP2-59778* at 1/1000, NBP2-67078* at 1/2000, GTX102072 at 1/3000, 3246** at 1/1000, A9188 at 1/30000, AC-PFN1-4** at 1/1000 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.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound? Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format? Yes Moreover, we do not score/recommend antibodies because we tested the antibodies under one set of conditions, and the scoring/recommendation is only valid under this precise experimental setup and in the cell line used. That being said, YCharOS reports serve as an invaluable guide pointing scientists to appropriate antibodies for their experimental needs.
Dr. Roy also asks whether the concentration units listed in Table 2 should be in mg/mL rather than μg/μl. To that we would like to elucidate the fact that both units of concentration are equivalent. Using mg/mL is equivalent to using μg/μl.
Thank you again to Partha Roy for your suggestions! Competing Interests: No competing interests were disclosed.
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