Identification of high-performing antibodies for Apolipoprotein E for use in Western Blot and immunoprecipitation

Apolipoprotein E is a secreted protein involved in mediating lipid distribution and metabolism among cells of specific tissues. The dysregulation of Apolipoprotein E can disturb cholesterol homeostasis, resulting in several diseases, including cardiovascular disease and Alzheimer’s disease. The therapeutic potential of Apolipoprotein E against these diseases demonstrates the importance of providing high-quality antibodies for this protein to the scientific community. In this study, we characterized fourteen Apolipoprotein E commercial antibodies for Western Blot and immunoprecipitation, 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
Apolipoprotein E, or APOE, is a 299 amino acid transcribed and secreted to regulate lipid homeostasis by controlling the uptake of cholesterol and lipoproteins via receptor-mediated endocytosis. 1,2Three major isoforms of APOE exist, apoE4, apoE3, and apoE2. 1 Despite only differing by single amino acid substitutions, their functionalities are altered at both the cellular and molecular levels. 1 Accordingly, the binding affinity of APOE to its ligand, the LDL receptor (LDLR), varies depending on the isoform. 2 ApoE3 and apoE4 bind to LDLR with high affinity while apoE2 binds with low affinity. 3][6][7] The critical role of APOE in health and disease highlights the need for additional research into the protein's mechanism of action and potential for therapeutic strategies. 8Mechanistic studies would be greatly facilitated with the availability of validated and high-quality antibodies.
Here, we compared the performance of a range of commercially-available antibodies for Apolipoprotein E and validated several antibodies for Western Blot and immunoprecipitation, enabling biochemical and cellular assessment of Apolipoprotein E properties and function.

Results and discussion
0][11] The first step was to identify a cell line(s) that expresses sufficient levels of Apolipoprotein E 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 have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655).Commercially available HAP1 cells expressed the APOE transcript at RNA levels above the average range of cancer cells analyzed.Parental and APOE knockout HAP1 cells were obtained from Horizon Discovery (Table 1).
Apolipoprotein E is predicted to be a secreted protein.Accordingly, we collected concentrated culture media from both WT and APOE KO cells and used the conditioned media to probe the performance of the antibodies (Table 2) side-by-side by Western Blot and immunoprecipitation. 10,11 The profiles of the tested antibodies are shown in Figures 1 and 2.
In conclusion, we have screened Apolipoprotein E commercial antibodies by Western Blot and immunoprecipitation and identified several high-quality antibodies under our standardized experimental conditions.The underlying data was previously uploaded to an open access repository, Zenodo. 12,13

Antibodies
All Apolipoprotein E antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly. 14All antibodies tested detect Human ApoE.Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from Thermo Fisher Scientific (cat.number 65-6520 and 62-6120).

REVISED Amendments from Version 2
In this new version, Figure 1 has been updated to repeat Western blot testing for antibodies ab1907* and MA5-15852* to determine whether they would successfully target ApoE when using a dilution of 1/200 rather than 1/1000.Table 1 was also updated.The title was also amended.Cell culture HAP1 WT and APOE KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 15  Western Blots were performed as described in our standard operating procedure. 16Midi precast 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat.number WXP42012BOX) were used and proteins were 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% bovine serum albumin (BSA) (Wisent, cat.number 800-095) in TBS with 0.1% Tween 20 (TBST) (Cell Signaling 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 with 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 on culture media Immunoprecipitation was performed as described in our standard operating procedure. 17Antibody-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 G -(for Mouse and rat antibodies) and protein A -(for rabbit antibodies) from Thermo Fisher Scientific (cat.number 10003D and 10002D, respectively).Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) was supplemented with the Halt Protease Inhibitor Cocktail 100X from Thermo Fisher Scientific (cat.number 78446) at a final concentration of 1Â.Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.Starved HAP1 WT culture media were concentrated as described above.0.6 mL aliquots at 1.5 mg/L of protein 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 precast midi 4-20% Tris-Glycine polyacrylamide gels.Prot-A: HRP (MilliporeSigma, cat.number P8651) was used as a secondary detection system at a concentration 0.4 μg/mL.

Valerio Leoni
University of Milano-Bicocca, Desio, Italy The manuscript Identification of high-performing antibodies for Apolipoprotein E for use in Western Blot and immunoprecipitation by Riham Ayoubi study the quality of commercial Anti-Apolipoporterin E antibodies.
The Apolipoprotein E is involved in lipid distribution and metabolism among cells, especially in the CNS.The dysregulation of Apolipoprotein E can disturb cholesterol homeostasis, resulting in several diseases, including cardiovascular disease and Alzheimer's disease.Provide high-quality antibodies for this protein to the scientific community is very important.They characterized fourteen Apolipoprotein E commercial antibodies for Western Blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls.
The study was well planned and interesting.than our pre-determined cut-off.An APOE knockout (KO) equivalent of the HAP1 cell line was purchased.Although the authors comprise of a team of experts in antibody characterization for the 3 tested applications, they are not ApoE experts and don't know all ApoE cleaved fragments.We do know that the signals which disappear in the KO line are ApoE species.If an ApoE researcher was interested in testing the antibodies for specific isoform, we would suggest creating or purchasing a KO line for the desired ApoE isoform and see whether the signal lost in the KO lysate compared to the WT.
The Western blot analysis was repeated for ab1907* and MA5-15852* where both antibodies were tested at 1/200 rather than 1/1000.The results and figure legend will be adjusted and a third version will be submitted to F1000.Please note that, to begin with, Western blot is not a recommended application for ab1907, which is why we did not increase the titration of the antibody in the original analysis.
For immunoprecipitation, the performance of each antibody is evaluated using an antibody that specifically immunodetected ApoE in the Western blot.The 13366* antibody was able to detect both of the most prominent ApoE isoforms with high specificity, which is why it was chosen for IP.
We hope we have answered all of your questions and concerns in this reply as well as the new version which has recently been submitted to F1000.
Competing Interests: No competing interests were disclosed.ApoE can be cleaved generating a C-terminal and a N-terminal peptide.The current work does not investigate whether the antibodies tested detect these cleavage products.Please state the immunogenic region to which the antibodies are raised in table 2.

2.
Please add details about species specificity to Table 2.If all detect human apoE please make this explicit.

3.
Cellular apoE exists as non-glycosylated and glycosylated isoforms, and secreted apoE is 4.
variably but predominantly glycosylated (refs 1-3).The authors have only shown detection of secreted apoE.It would be helpful to show the ability of the antibodies to detect cellular apoE glycoforms.This will also indicate whether the antibodies display non-specific binding to other cellular proteins.
Provide full name for HAP1 before abbreviation is used.

5.
What is the apoE genotype of the HAP1 cell line they have used?6.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com Wb=Western Blot; IF=immunofluorescence; IP=immunoprecipitation. *Monoclonal antibody.**Recombinant antibody.

Figure 2 .
Figure2.Apolipoprotein E antibody screening by immunoprecipitation on culture media.Immunoprecipitation was performed on 0.9 mg concentrated culture media from HAP1 WT, and using 2.0 μg of the indicated Apolipoprotein E antibodies pre-coupled to protein G or protein A magnetic beads.Samples were washed and processed for Western Blot with the indicated Apolipoprotein E antibody.Antibody 13366** was used at 1/500 for all Western Blots.The Ponceau stained transfers of each blot are shown.SM=3% starting material; UB=3% unbound fraction; IP=immunoprecipitate; HC=heavy chain; *Monoclonal antibody, **Recombinant antibody.

Table 1 .
Summary of the cell lines used.

Table 2 .
Summary of the Apolipoprotein E antibodies tested.

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
Could the authors explain why they chose 13366** for western blotting after IP? Did they use other antibodies for this purpose, if yes, were the results similar? 4.
Results are clear, Figure and table informative A minor linguistic revision is recommended

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

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.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 1
Reviewer Report 19 July 2023 https://doi.org/10.5256/f1000research.146914.r186248 1.