A guide to selecting high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence

Rab1 is a highly conserved small GTPase that exists in humans as two isoforms: Rab1A and Rab1B, sharing 92% sequence identity. These proteins regulate vesicle trafficking between the endoplasmic reticulum (ER) and Golgi and within the Golgi stacks. Rab1A and Rab1B may be oncogenes, as they are frequently dysregulated in various human cancers. Moreover, they contribute to the progression of Parkinson’s disease. The availability of high-quality antibodies specific for Rab1A or Rab1B is essential to understand the distinct functions of these Rab1 proteins in both health and diseaseand to enhance the reproducibility of research involving these proteins. In this study, we characterized seven antibodies targeting Rab1A and five antibodies targeting Rab1B for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a much larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a valuable resource for the scientific community. While uses of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.


Introduction
Multiple steps in membrane trafficking are coordinated by Rab proteins, a family of small guanosine triphosphatases (GTPase). 12][3] When activated, Rab proteins partake in crosstalk through shared effector proteins or through Rab activators to ensure vesicle traffic is spatiotemporally regulated. 1Homologous to YTP1 in yeast, the Rab1 human proteins play key roles in regulating ER-Golgi and intra-Golgi transport.They exist as two isoforms in humans, Rab1A and Rab1B.5][6] Rab1B has been proposed to function in the initial stages of the secretory pathways, serving to assemble and disassemble machinery required for vesicle fission and fusion, 4 whereas Rab1A exhibits unique functions such as its involvement in cell adhesion and migration, and plays a role in facilitating autophagosome formation, an early step in the autophagy pathway 7,8 Elevated expression of RAB1A and RAB1B genes have implications in various cancer types, including colorectal cancer, 9 hepatocellular cancer, 10 gliomas, 11 tongue carcinomas, prostate cancer 12 for RAB1A and colorectal cancer, 13 hepatocellular cancer, 14 and prostate cancer 12 for RAB1B.Rab1A in human cancer is highly studied in comparison to Rab1B as abnormal expression of Rab1A activates mTORC1 signalling, promoting tumour growth, invasion and ultimately cancer progression. 9Rab1 proteins are also involved in the pathogenesis of Parkinson's disease, characterized by accumulation of α-synuclein.Inhibition of ER-Golgi traffic has been reported to trigger α-synuclein aggregation, suggesting that an increase in production of Rab1 proteins can potentially rescue this α-synuclein toxic phenotype. 15Further research is required to understand the role of Rab1A and Rab1B in various diseased states and their potential as therapeutic targets to slow the progression of cancer and neurodegeneration.In-depth mechanistic investigations would significantly benefit from the accessibility of high-performing antibodies, which can help elucidate the underlying processes and pathways involving Rab1A and Rab1B.An editorial by Biddle et al. can provide valuable insights on how to interpret the antibody characterization data found in this article. 168][19] Here, twelve commercially available antibodies that target either Rab1A or Rab1B were tested in Western Blot, immunoprecipitation and immunofluorescence applications using a knockout-based validation approach.This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of Rab1A and Rab1B properties and function.

Results and discussion
Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. 20,21The first step was to identify a cell line(s) that expresses sufficient endogenous levels of a given protein to generate a measurable signal.To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the Rab1 isoforms at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655).Commercially available HAP1 cells express Rab1A and Rab1B transcripts at RNA levels above the average range of cancer cells analyzed.Parental and RAB1A and RAB1B KO HAP1 cells were obtained from Horizon Discovery (Table 1).
For Western Blot experiments, we resolved proteins from WT and RAB1A and RAB1B KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1). 21Figure 1 indicates which antibodies are intended for Rab1A (A) or Rab1B (B).In the results, it was observed that Rab1A antibodies, namely ab302545**, NBP3-11042*, NBP3-11043*, 13075** and 11671-1-AP immunodetected their target, Rab1A protein as a ~23 kDa band in the HAP1 WT lysate while the levels of Rab1A increased by ~2-3 fold in the lysates of Rab1B KO cells.Similarly, Rab1B antibodies 17824-1-AP

REVISED Amendments from Version 1
The title of the article has been changed to better reflect the nature of this Data Note which presents antibody characterization data for Rab1A and Rab1B.The purpose of our Data Notes are to guide readers in selecting high-performing antibodies, but we do not provide data analysis nor recommendations, as the authors wish to remain unbiased.Any further responses from the reviewers can be found at the end of the article and PA5-77240 detect Rab1B at ~23 kDa in the HAP1 WT lysate, and revealed a similar ~2-3 fold increase in Rab1B protein level in the Rab1A KO lysate.These results demonstrated that a compensatory mechanism exists to ensure that overall Rab1 protein levels remain balanced.As per our standard procedure, we next used the antibodies to immunoprecipitateRab1A and Rab1B from HAP1 cell extracts.The performance of each antibody was evaluated by detecting the Rab1A and Rab1B protein in extracts, in the immunodepleted extracts and in the immunoprecipitates using an antibdy that was validated by Western Blot (Figure 2). 21r immunofluorescence, antibodies were screened using a mosaic strategy, as per our standard procedure.First, the HAP1 WT and RAB1A KO cells were plated together in the same tissue culture wells, using different colour fluorescent dyes to distinguish the two cell lines, and the seven Rab1A antibodies were tested.Then, HAP1 WT and RAB1B KO cells were plated together using the same strategy, and the five Rab1B antibodies were tested.Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).Quantification of immunofluorescence intensity hundreds of WT and KO cells was performedfor each antibody tested.The images presented in Figure 3 are representative of the results of this analysis.
In conclusion, we have screened seven Rab1A and Rab1B commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence.Several high-quality antibodies that selectively detect either Rab1A or Rab1B under the standardized experimental conditions were identified in each of the tested applications.In our efforts to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming be removed from the commercial antibody market.However, the authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocols -any conclusions remain relevant within the confines of the experimental setup and cell line used in this study.Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles.This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.
The underlying data for this study can be found on Zenodo, an open access repository for which YCharOS has its own community. 22,23

Antibodies
All Rab1A and Rab1B antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 24Peroxidase-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 antimouse secondary antibodies are from Thermo Fisher Scientific (cat.number A21429 and A21424).

Cell culture
Both HAP1 WT and RAB1A and RAB1B KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 25

Antibody screening by immunoprecipitation
Immunoprecipitation was performed as described in our standard operating procedure.Antibody-bead conjugates were prepared by adding 2 μg or 10 μL of antibody NBP3-18251 (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).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) supplemented with protease inhibitor.Lysates were rocked for 30 min at 4°C and spun at 110,000 Â 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 10% Bis-Tris polyacrylamide gels.Prot-A: HRP (MilliporeSigma, cat.number P8651) was used as a secondary detection system at a concentration of 0.3 μg/mL.

Antibody screening by immunofluorescence
Immunofluorescence was performed as described in our standard operating procedure. 21HAP1 WT and the HAP1 RAB1A and RAB1B KO cell lines 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 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. 26 In this report, Moleon and colleagues characterize several antibodies directed against the human Rab1A or Rab1B proteins.Rab1 proteins are small GTP-binding proteins that participate in vesicle trafficking, among other cellular functions.Interest in these proteins also stems from their potential role as oncogenes, as several cancer types have been shown to upregulate Rab1A or Rab1B.The authors carefully characterized 7 Rab1A and 5 Rab1B antibodies by western blot, immunoprecipitation, and immunofluorescence assays using KO and isogenic parental cell lines.The results are clearly presented, and the methods detailed enough to allow replication.Overall, there are no specific concerns in my view.

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Only few minor typos were identified such as missing spaces in the abstract ("diseaseand") and on page 5 ("immunoprecipitateRab1A", "antibdy", and "performedfor").3.While "the authors recommend the antibodies that demonstrated to be underperforming be removed from the commercial antibody market", one cannot conclude usefulness of these antibodies in cell lines derived from other species such as mouse, hamster (CHO cells) and rat.

Is the rationale for creating the dataset(s) clearly described? Partly
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 Competing Interests: No competing interests were disclosed.
Reviewer Expertise: cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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Figure 3
Figure 3 has also been updated.

Figure 2 .
Figure 2. Rab1A and Rab1B antibody screening by immunoprecipitation.HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated Rab1A and Rab1B antibodies pre-coupled to Dynabeads protein A or protein G.Samples were washed and processed for Western Blot with the indicated Rab1A and Rab1B antibodies.Figure 2A represents the IP results for antibodies intended to immunodetect Rab1A while Figure 2B represents the results for antibodies intended to immunodetect Rab1B.For Rab1A Western Blots (A), ab302545** was used at 1/200.For Rab1B Western Blots (B), 17824-1-AP was used at 1/500.The Ponceau stained transfers of each Blot are shown.SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; LC=antibody light chain.*Monoclonal antibody, **Recombinant antibody.
Figure 2. Rab1A and Rab1B antibody screening by immunoprecipitation.HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated Rab1A and Rab1B antibodies pre-coupled to Dynabeads protein A or protein G.Samples were washed and processed for Western Blot with the indicated Rab1A and Rab1B antibodies.Figure 2A represents the IP results for antibodies intended to immunodetect Rab1A while Figure 2B represents the results for antibodies intended to immunodetect Rab1B.For Rab1A Western Blots (A), ab302545** was used at 1/200.For Rab1B Western Blots (B), 17824-1-AP was used at 1/500.The Ponceau stained transfers of each Blot are shown.SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; LC=antibody light chain.*Monoclonal antibody, **Recombinant antibody.

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Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Cell biology and neuroscienceI confirm that I have read this submission and believe that I have an appropriate level of2.A limitation of this study is that the authors analyzed only the human cell line HAP1 and not widely used cells such as Hela cells and 293 cells.

Table 1 .
Summary of the cell lines used.
1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat.number89901) supplemented with 1Â protease inhibitor cocktail mix (MilliporeSigma, cat.numberP8340).Lysates were sonicated briefly and incubated for 30 min on ice.Lysates were spun at ~110,000 Â 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.numberPM008-0500) was used.Western Blots were performed with a precast midi 10% Bis-Tris polyacrylamide gels from 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 1Â sample reducing agent (Thermo Fisher Scientific, cat.number NP0009) 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).Membranes incubated with primary antibodies NBP3-11043*, 13075**, PA5-44578, PA5-104066, NBP3-18251 and PA5-104067 were developed with Clarity Western ECL Substrate, and the remaining antibodies with Pierce ECL.
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).