APBB1 Antibody

What molecular weight should I expect when detecting APBB1 in Western blot experiments?

APBB1 exhibits interesting molecular weight variations that researchers should be aware of. While the calculated molecular weight is approximately 77 kDa (708 amino acids), the observed molecular weight often differs:

For optimal detection, use positive controls such as brain tissue lysates (mouse, rat, or pig) where APBB1 is highly expressed. When troubleshooting, remember that different antibodies may preferentially detect specific isoforms or modified forms of APBB1 .

In which tissues and cell types is APBB1 most highly expressed?

APBB1 shows distinct tissue-specific expression patterns:

• High expression: Brain tissue, particularly cerebellum, hippocampus, and cortex

• Validated cell lines: SH-SY5Y (neuroblastoma), U-87 MG (glioblastoma), U2OS, and A549 cells

For immunohistochemistry applications, mouse cerebellum and general brain tissue are recommended as positive controls . When designing experiments targeting APBB1, these tissues and cell lines serve as reliable positive controls for antibody validation .

What are the recommended applications for different types of APBB1 antibodies?

Different APBB1 antibodies are optimized for specific applications:

Always titrate the antibody concentration in your specific experimental system for optimal results, as tissue preparation methods and detection systems can significantly impact performance .

How can I optimize antigen retrieval for APBB1 immunohistochemistry in brain tissue?

Antigen retrieval is critical for successful APBB1 detection in fixed tissues. Based on validation data:

• Primary recommendation: TE buffer at pH 9.0 provides optimal antigen unmasking

• Alternative method: Citrate buffer at pH 6.0 may also be effective but typically yields lower signal intensity

For mouse brain tissue sections, the following protocol yields consistent results:

• Deparaffinize and rehydrate tissue sections using standard procedures

• Perform heat-induced epitope retrieval using TE buffer (pH 9.0) for 15-20 minutes

• Allow sections to cool to room temperature for 20 minutes before proceeding with blocking steps

• Continue with standard IHC procedures using recommended antibody dilutions (1:50-1:500)

This optimization is particularly important for detecting APBB1 in cerebellar sections, where proper antigen retrieval significantly enhances detection sensitivity .

What explains the discrepancy between calculated and observed molecular weights of APBB1?

The discrepancy between calculated (77 kDa) and observed (65-100 kDa) molecular weights of APBB1 stems from several factors:

• Post-translational modifications: Phosphorylation significantly increases the apparent molecular weight to approximately 100 kDa

• Proteolytic processing: The major isoform (p97FE65) can be converted to a 65 kDa N-terminal fragment in certain cellular contexts

• Alternative splicing: Different isoforms may present different electrophoretic mobility patterns

Research by Proteintech demonstrates that phosphorylation-specific bands appear around 100 kDa, while the 55-60 kDa bands sometimes observed represent FE65-like proteins that show cross-reactivity with some antibodies . When interpreting Western blot results, researchers should carefully consider which form of APBB1 is most relevant to their experimental question .

How can APBB1 antibodies be used to study the interaction between APBB1 and APP in Alzheimer's disease research?

APBB1 (Fe65) interacts with APP through specific protein domains:

• The interaction primarily occurs between APBB1's C-terminal phosphotyrosine interaction domain (PID2) and the APP intracellular domain (AICD)

• APBB1 contains three major protein-protein interaction domains: one WW domain and two phosphotyrosine interaction domains (PID)

For studying this interaction, researchers can employ:

• Co-immunoprecipitation: Using APBB1 antibodies (0.5-4.0 μg for 1.0-3.0 mg of protein lysate) to pull down complexes from brain tissue, followed by Western blot analysis for APP

• Immunofluorescence co-localization: Using fluorescently-labeled APBB1 antibodies like CL488-67077 (1:50-1:500 dilution) to visualize subcellular co-localization with APP in neuronal models or brain tissue sections

• Proximity ligation assays: For detecting direct protein-protein interactions in situ with high specificity

These approaches help elucidate the functional consequences of APBB1-APP interactions in Alzheimer's disease pathogenesis and potential therapeutic interventions .

What is the significance of APBB1 as a biomarker in ACPA-negative rheumatoid arthritis?

Recent genome-wide autoantibody screening identified anti-APBB1 as one of five promising biomarkers for ACPA-negative rheumatoid arthritis (RA):

The significance of anti-APBB1 is particularly noteworthy in early-stage ACPA-negative RA, where conventional diagnostic markers are often insufficient. In patients with disease duration <2 years, anti-APBB1 positivity reached 56.6%, compared to established stage RA .

These findings suggest that incorporating anti-APBB1 testing into diagnostic panels could significantly improve early detection of ACPA-negative RA, addressing a critical unmet clinical need. A multi-marker diagnostic model incorporating these autoantibodies achieved 23.8% true positive rate in ACPA-negative RA patients, offering potential improvements over single-marker approaches .

How should APBB1 antibodies be validated for specificity in clinical biomarker studies?

For clinical biomarker applications, rigorous antibody validation is essential:

• Cross-reactivity assessment: Test against a panel of related proteins to ensure specificity

• Multiple tissue validation: Confirm reactivity in tissues known to express APBB1 (brain) and those that don't

• Multiple technique validation: Verify consistent results across different detection methods (WB, IHC, IF, ELISA)

• Blocking peptide controls: Use immunogen peptides to confirm binding specificity

• Knockout/knockdown controls: If available, use APBB1-knockout tissues or cells as negative controls

For autoantibody biomarker studies specifically, validation requires:

• Testing against large cohorts of patients with related autoimmune conditions to establish disease specificity

• Correlation with clinical parameters and disease progression

• Comparison with established biomarkers (e.g., ACPA)

The HuProt array approach demonstrated in the RA biomarker study provides a systematic validation method, where anti-APBB1 showed 27.1% sensitivity and 87.7% specificity in ACPA-negative RA patients .

What storage and handling conditions are critical for maintaining APBB1 antibody performance?

Proper storage significantly impacts antibody longevity and performance:

For lyophilized antibodies, reconstitute in the recommended volume of distilled water or buffer to achieve the specified concentration (typically 0.5-1 mg/ml) . Some antibody preparations contain carrier proteins like BSA (0.1%) to enhance stability .

The CoraLite® Plus 488-conjugated APBB1 antibody requires additional protection from light exposure to prevent fluorophore degradation .

How can I troubleshoot weak or non-specific bands when using APBB1 antibodies in Western blot?

When encountering issues with APBB1 detection:

• For weak signals:

  • Increase antibody concentration within recommended range (1:1000-1:6000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence detection systems

  • Increase protein loading (50-100 μg of total protein for brain tissue)

• For non-specific bands:

  • Be aware that APBB1 can appear at different molecular weights (65 kDa, 97-100 kDa)

  • A non-specific band at 55-60 kDa may represent FE65-like protein

  • Use positive controls (mouse/rat brain tissue) for band identification

  • Optimize blocking conditions (5% non-fat milk or BSA)

  • Increase washing stringency with additional TBST washes

• For tissue-specific optimization:

  • Brain tissue: A549 cells, SH-SY5Y cells, U-87 MG cells, U2OS cells, and mouse/rat brain tissue are validated positive controls

  • Decrease antibody concentration for tissues with high APBB1 expression

Remember that observed molecular weights may differ from calculated values due to post-translational modifications and isoform expression .

What protocols maximize signal-to-noise ratio when using fluorescent-conjugated APBB1 antibodies?

For optimal results with fluorescent conjugates like CoraLite® Plus 488-APBB1:

• Tissue preparation:

  • Use fresh frozen sections or properly fixed paraffin-embedded tissues

  • For brain tissue, optimal fixation is 4% paraformaldehyde for 24 hours

  • Perform antigen retrieval with TE buffer pH 9.0 for highest signal recovery

• Blocking optimization:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Add 0.3% Triton X-100 for permeabilization in brain tissue sections

  • Include 0.1% sodium azide to prevent microbial growth during longer incubations

• Antibody dilution:

  • Start with 1:200 dilution and titrate as needed (range: 1:50-1:500)

  • Extend incubation time to overnight at 4°C for maximum sensitivity

  • Prepare antibody in buffer containing 2% serum to reduce background

• Imaging considerations:

  • CoraLite® Plus 488 has excitation/emission maxima of 493/522 nm

  • Include appropriate controls for autofluorescence, especially in brain tissue

  • Use DAPI counterstaining for nuclei visualization

  • Apply antifade mounting medium to prevent photobleaching

For mouse brain tissue, positive signal should be detected in cerebellum, hippocampus, and cortex regions, offering internal positive controls for protocol optimization .

How can APBB1 antibodies contribute to DNA damage response research?

APBB1 plays a critical role in DNA damage response beyond its association with APP:

• It translocates to the nucleus upon DNA damage and may contribute to apoptotic signaling

• APBB1 specifically recognizes and binds histone H2AX phosphorylated on Tyr-142 (H2AXY142ph) at double-strand breaks (DSBs)

• It potentially recruits pro-apoptotic factors like MAPK8/JNK1

Research applications include:

• Using APBB1 antibodies to track nuclear translocation following DNA damage

• Co-localization studies with γH2AX to identify DSB repair complexes

• Immunoprecipitation with APBB1 antibodies to identify novel binding partners in DNA repair pathways

This expanding research area suggests APBB1's multiple cellular functions extend beyond its established role in Alzheimer's disease, opening new avenues for investigation in cancer biology and cellular stress response mechanisms .

What is the significance of proteolytic processing of APBB1 and how can different antibodies detect these events?

APBB1 undergoes important proteolytic processing that affects its function:

• The major isoform of FE65 (97-kDa p97FE65) can be converted to a 65-kDa N-terminal fragment through proteolytic cleavage

• This processing may regulate APBB1's interaction with APP and other binding partners

• Different cleavage events may direct APBB1 to different subcellular compartments

For detecting specific forms:

• Antibodies targeting different epitopes can selectively detect full-length versus processed forms

• C-terminal-specific antibodies (like abx269960) detect only full-length APBB1

• N-terminal-specific antibodies (like ARP55471_P050) can detect both forms

• Western blotting with antibodies targeting different regions can map cleavage events

Understanding proteolytic processing of APBB1 provides insights into regulatory mechanisms controlling APP processing and potentially influencing amyloid-beta production in Alzheimer's disease pathogenesis .