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Data Note

Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence

[version 1; peer review: 1 approved, 1 approved with reservations]
PUBLISHED 09 Aug 2023
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This article is included in the Cell & Molecular Biology gateway.

This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.

Abstract

The amyloid-beta precursor protein is a transmembrane protein expressed in many tissues and highly concentrated in the brain. The protein is of significant interest due to its involvement in the generation of amyloidogenic β-amyloid peptides, prone to plaque formation that is characteristic of Alzheimer’s Disease. The scientific community would benefit from the availability of high-quality anti-amyloid-beta precursor protein antibodies to enhance reproducible research on this target. In this study, we characterized eleven amyloid-beta precursor protein commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence 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.

Keywords

Uniprot ID P05067, APP, Amyloid-beta precursor protein, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence

Introduction

The amyloid-beta precursor protein, encoded by the APP gene, is a transmembrane protein with a single transmembrane domain, a large extracellular ectodomain and a short cytoplasmic tail.1 Although ubiquitously expressed, amyloid-beta precursor protein is predominantly found on the surface of neurons, where it promotes neurite growth, neuronal adhesion and axonogenesis.2

Discovered and isolated in the 1980’s, amyloid-beta precursor protein has become a significant area of interest as its proteolytic cleavage can give rise to amyloid β-proteins (Aβ).1,3,4 Aβ are small 40-42 amino acid-long peptides related to the pathogenesis of Alzheimer’s Disease as their accumulation can result in the formation of senile amyloid plaques.1,5 Further research is required to elucidate the pathway underlying the proteolytic processing of amyloid-beta precursor protein and in turn determine its potential as a diagnostic marker or therapeutic target to prevent degenerative brain progression. Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.

In this study, we compared the performance of a range of commercially available antibodies for amyloid-beta precursor protein and identified high-performing antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of amyloid-beta precursor protein’s properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.616 The first step was to identify a cell line(s) that expresses sufficient 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 target at levels greater than 2.5 log2 (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 expressed the amyloid-beta precursor protein transcript at RNA levels above the average range of cancer cells analyzed. Parental and APP KO HAP1 cells were obtained from Horizon Discovery (Table 1).

Table 1. Summary of the cell lines used.

InstitutionCatalog numberRRID (Cellosaurus)Cell lineGenotype
Horizon DiscoveryC631CVCL_Y019HAP1WT
Horizon DiscoveryHZGHC005368c010CVCL_SD04HAP1APP KO

For Western Blot experiments, we resolved proteins from WT and APP KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).716

dc1d235b-2c03-4d74-bfef-6bcb57409875_figure1.gif

Figure 1. Amyloid-beta precursor protein antibody screening by Western Blot.

Lysates of HAP1 (WT and APP KO) were prepared and 50 μg of protein were processed for Western Blot with the indicated Amyloid-beta precursor protein antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies ab126732**, 29765** and 76600** which were all titrated to 1/500, as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: ab126732** at 1/500, ab252814** at 1/500, ab252816** at 1/500, ARP33075 at 1/500, ARP34012 at 1/500, 29765** at 1/500, 76600** at 1/500, 25524-1-AP at 1/500, 13-0200* at 1/200, 14-9749-80* at 1/500, MA5-35187** at 1/500. Predicted band size: 87 kDa. *Monoclonal antibody, **Recombinant antibody.

For immunoprecipitation experiments, we used the antibodies to immunopurify amyloid-beta precursor protein from HAP1 cell extracts. The performance of each antibody was evaluated by detecting amyloid-beta precursor protein protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).716

dc1d235b-2c03-4d74-bfef-6bcb57409875_figure2.gif

Figure 2. Amyloid-beta precursor protein antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated Amyloid-beta precursor protein antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated Amyloid-beta precursor protein antibodies. For Western Blot, MA5-35187** and ab252816** were used at 1/500. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate, *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, antibodies were screened using a mosaic strategy.17 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).

dc1d235b-2c03-4d74-bfef-6bcb57409875_figure3.gif

Figure 3. Amyloid-beta precursor protein antibody screening by immunofluorescence.

HAP1 WT and APP KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with an optically clear flat-bottom. Cells were stained with the indicated Amyloid-beta precursor protein antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, we tested antibodies at 1/100 and 1/500. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab126732** at 1/500, ab252814** at 1/500, ab252816** at 1/500, ARP33075 at 1/500, ARP34012 at 1/500, 29765** at 1/100, 76600** at 1/50, 25524-1-AP at 1/500, 13-0200* at 1/500, 14-9749-80* at 1/500, MA5-35187** at 1/1000. *Monoclonal antibody, **Recombinant antibody, Bars=10 μm.

In conclusion, we have screened eleven amyloid-beta precursor protein commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions. The underlying data can be found on the Zenodo open access repository.18,19

Methods

Antibodies

All amyloid-beta precursor protein antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.20 Peroxidase-conjugated goat anti-mouse, anti-mouse and anti-rat antibodies are from Thermo Fisher Scientific (cat. number 62-6520, 65-6120 and 31470, respectively). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429).

Table 2. Summary of the amyloid-beta precursor protein antibodies tested.

CompanyCatalog numberLot numberRRID (Antibody Registry)ClonalityClone IDHostConcentration (μg/μL)Vendors recommended applications
Abcamab126732**GR34478901AB_11131727recombinant-monoEPR5118-34rabbit0.114Wb
Abcamab252814**GR34013051AB_29252241recombinant-mono14D6rat1.154Wb
Abcamab252816**GR33342983AB_29252231recombinant-mono2E9rat0.583Wb, IP
Aviva Systems BiologyARP33075QC3561-90401AB_2044934polyclonal-rabbit0.5Wb
Aviva Systems BiologyARP34012QC47046-42571AB_2044933polyclonal-rabbit0.5Wb
Cell Signaling Technology29765**1AB_29252211recombinant-monoE8B3Orabbitn/aWb, IP
Cell Signaling Technology76600**1AB_29252221recombinant-monoE4H1Urabbitn/aWb, IP
Proteintech25524-1-AP51555AB_2880118polyclonal-rabbit0.55Wb, IF
Thermo Fisher Scientific13-0200*XC339604AB_2532993monoclonalLN27mouse0.5Wb, IP, IF
Thermo Fisher Scientific14-9749-80*2484403AB_2572977monoclonal22C11mouse0.5Wb, IF
Thermo Fisher ScientificMA5-35187**XH3670292AB_2849091recombinant-monoARC0465rabbit0.93Wb, IF

* Monoclonal antibody.

** Recombinant antibody.

1 Refers to RRID recently added to the Antibody Registry (in May 2023), they will be available on the Registry website in coming weeks.

Cell culture

Both HAP1 WT and APP KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.21 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.22 HAP1 WT and APP 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 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 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer (bio-Rad, cat. number 1610772), loaded in Laemmli loading sample buffer (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 was scanned to show together with individual Western blot. Blots were blocked with 5% milk for 1 h, 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 h at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat. number 32106) prior to detection with the iBright CL1500 Imaging System (Thermo Fisher Scientific, cat. number A44240).

Antibody screening by immunoprecipitation

Immunoprecipitation was performed as described in our standard operating procedure.23 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 (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 and rat antibodies) (Thermo Fisher Scientific, cat. number 10002D and 10004D, respectively). Tubes were rocked for ~2 h 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 × 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 ~2 h 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.

Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure.717 HAP1 WT and APP KO were labelled with a green and a deep red fluorescence dye, respectively (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 on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37°C, 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% 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 Amyloid-beta precursor protein 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 h 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 20x/0.95 NA 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 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.24 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).

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Ayoubi R, Fotouhi M, Worrall D et al. Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 12:956 (https://doi.org/10.12688/f1000research.139867.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
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Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
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Reviewer Report 13 Feb 2024
Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany 
Approved
VIEWS 16
This manuscript "Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence" fulfils the criteria for publication as a Data Note. The scientific topic - the immunological detection of the amyloid precursor protein APP ... Continue reading
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Rahfeld JU. Reviewer Report For: Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 12:956 (https://doi.org/10.5256/f1000research.153179.r237210)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 24 Jan 2024
Amal Kaddoumi, Auburn University, Auburn, Alabama, USA 
Approved with Reservations
VIEWS 23
In this report, the authors compared eleven APP commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol using HAP1-APP expressing cells (WT) and HAP1-APP KO cells. This report could be useful if the authors elaborated more ... Continue reading
CITE
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HOW TO CITE THIS REPORT
Kaddoumi A. Reviewer Report For: Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 12:956 (https://doi.org/10.5256/f1000research.153179.r237217)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 13 Apr 2024
    Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada
    13 Apr 2024
    Author Response
    Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 13 Apr 2024
    Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada
    13 Apr 2024
    Author Response
    Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that ... Continue reading

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Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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