ACP2 Antibody

What is ACP2 and why are antibodies against it important in research?

ACP2 (Acid Phosphatase 2) is a lysosomal enzyme with a calculated molecular weight of approximately 48 kDa, though it may appear at different sizes (52-76 kDa) in various tissues and cell types due to post-translational modifications . This protein plays a crucial role in membrane fusion during influenza virus entry, making it an important target for antiviral research .

Antibodies against ACP2 are vital research tools that enable the detection, quantification, and characterization of this protein in various experimental systems. They facilitate investigations into the protein's expression patterns across different tissues, subcellular localization, and functional roles in both normal physiological processes and disease contexts . Recent studies have identified ACP2 as necessary for influenza virus replication, highlighting the importance of reliable antibodies for studying this protein in infectious disease research .

What are the key specifications to consider when selecting an ACP2 antibody?

When selecting an ACP2 antibody for research, several critical specifications should be evaluated:

• Reactivity spectrum: Confirm the antibody's validated reactivity with your species of interest. Available ACP2 antibodies commonly react with human, mouse, and rat samples .

• Applications compatibility: Verify the antibody has been validated for your intended application. ACP2 antibodies are available for various applications including Western Blot (WB), ELISA, and Immunofluorescence (IF/ICC) .

• Clonality: Consider whether a polyclonal or monoclonal antibody better suits your research needs. Polyclonal antibodies offer higher sensitivity by recognizing multiple epitopes, while monoclonal antibodies provide higher specificity .

• Host species: Select an antibody raised in a species that avoids cross-reactivity issues with your experimental system .

• Expected band size: ACP2 may appear at different molecular weights depending on the tissue or cell type. Common observed sizes include 48 kDa (calculated), 52 kDa, 63 kDa, and 76 kDa .

• Storage requirements: Most ACP2 antibodies require storage at -20°C for long-term stability, with specific handling instructions after reconstitution .

Thoroughly examining these specifications ensures selection of an appropriate antibody that will yield reliable, reproducible results in your experimental system.

How should ACP2 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of ACP2 antibodies are essential for maintaining their activity and ensuring consistent experimental results. According to manufacturer guidelines, the following practices are recommended:

• Long-term storage: Store lyophilized ACP2 antibodies at -20°C for up to one year from the date of receipt . This temperature preserves antibody integrity by minimizing degradation and maintaining binding affinity.

• After reconstitution: Once reconstituted, ACP2 antibodies can be stored at 4°C for approximately one month . For longer storage periods, aliquot the reconstituted antibody and store at -20°C for up to six months.

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces antibody activity. Prepare small, single-use aliquots before freezing to minimize the number of freeze-thaw cycles .

• Working dilutions: Prepare working dilutions immediately before use. For Western blot applications, typical dilutions range from 1:500 to 1:1000, while immunofluorescence applications may require dilutions between 1:10 and 1:100 .

• Transport conditions: During transportation between laboratories, maintain cold chain integrity using appropriate cold packs or dry ice.

Adhering to these storage and handling guidelines will help preserve antibody function and extend shelf life, ultimately improving research reproducibility and data quality.

What methodological approaches can resolve the discrepancy between the calculated and observed molecular weights of ACP2?

The discrepancy between the calculated molecular weight of ACP2 (48 kDa) and its observed weights (52-76 kDa) presents an interesting research challenge . Several methodological approaches can be employed to investigate this phenomenon:

• Deglycosylation experiments: Treat protein samples with enzymes like PNGase F or EndoH before Western blotting to determine if glycosylation accounts for the higher observed weights. A shift to lower molecular weight bands after treatment would confirm glycosylation as a contributing factor.

• Phosphatase treatment: Incubate samples with lambda phosphatase to investigate whether phosphorylation contributes to the observed size differences.

• 2D gel electrophoresis: Separate proteins by both isoelectric point and molecular weight to identify potential post-translationally modified ACP2 isoforms.

• Mass spectrometry analysis: Employ LC-MS/MS analysis on immunoprecipitated ACP2 to precisely characterize post-translational modifications and identify specific modified residues.

• Comparison across different tissues: The observed molecular weight of ACP2 varies among different tissues and cell lines. For instance, Western blot analyses show bands at approximately:

  • 52 kDa in human Hela, U251, MCF-7, Caco-2, 293T, A549, and SH-SY5Y cell lysates

  • 52 kDa in rat brain tissue, rat C6 cells, mouse brain tissue, and mouse Neuro-2a cells

  • 76 kDa in human T-47D, Hela, U251, and RT4 cells, as well as rat and mouse liver and pancreas tissues

  • 63 kDa and 52 kDa in mouse kidney tissue, HepG2 cells, HL-60 cells, and mouse brain tissue

This tissue-specific variation suggests potential regulation through alternative splicing or tissue-specific post-translational modifications, which could be explored through tissue-comparative analyses.

By systematically applying these approaches, researchers can elucidate the molecular basis for the observed weight differences and gain insights into the potential functional consequences of these modifications.

How can researchers validate the specificity of ACP2 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable research outcomes. For ACP2 antibodies, several complementary approaches can be implemented:

• Knockdown/knockout validation: One of the most rigorous approaches involves depleting ACP2 expression through siRNA knockdown or CRISPR-Cas9 knockout, followed by immunoblotting. This approach has been demonstrated in influenza research, where siACP2 effectively reduced endogenous ACP2 protein levels in both non-infected and infected cells, confirming antibody specificity .

• Multiple antibody comparison: Compare detection patterns using multiple antibodies targeting different epitopes of ACP2. Consistent detection patterns would support antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before probing samples. Disappearance of specific bands confirms that the antibody is binding to its intended target.

• Recombinant protein positive control: Include purified recombinant ACP2 protein alongside experimental samples to confirm correct band identification.

• Cross-species reactivity analysis: Compare detection patterns across validated reactive species (human, mouse, rat) to identify conserved binding patterns . Consistent detection at expected molecular weights across evolutionarily related species provides evidence for specificity.

• Tissue expression profiling: Compare antibody detection patterns with known tissue expression profiles of ACP2. For example, validation data shows consistent detection of ACP2 in brain, liver, pancreas, and kidney tissues, aligning with known expression patterns .

What experimental considerations are important when using ACP2 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) studies with ACP2 antibodies require careful experimental design to ensure successful protein complex isolation while minimizing artifacts. Key considerations include:

• Antibody selection: Choose an ACP2 antibody specifically validated for immunoprecipitation applications. Polyclonal antibodies often perform better for Co-IP due to their recognition of multiple epitopes, increasing the probability of capturing native protein complexes .

• Lysis buffer optimization: ACP2 is a lysosomal protein, requiring careful buffer selection to maintain protein-protein interactions while effectively solubilizing membrane components. Consider testing multiple lysis conditions:

  • NP-40 or Triton X-100 (0.5-1%) for milder extraction

  • RIPA buffer for more stringent conditions

  • Include protease and phosphatase inhibitors to prevent degradation

• Cross-linking considerations: For transient or weak interactions, consider using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) before cell lysis to stabilize protein complexes.

• Control experiments:

  • Include IgG control from the same species as the ACP2 antibody

  • Perform parallel Co-IP with lysates from ACP2-depleted cells (siRNA or CRISPR knockout) as a specificity control

  • Consider using tagged-ACP2 overexpression systems for validation of novel interactions

• ACP2 complexes detection: Based on Western blot validation data, ACP2 displays molecular weight variability (52-76 kDa) across different tissues and cell types . Ensure blotting conditions can detect this range when analyzing Co-IP samples.

• Validation of interactions: Confirm novel interactions through reciprocal Co-IP and complementary techniques such as proximity ligation assay (PLA) or FRET (Fluorescence Resonance Energy Transfer).

These considerations help address the unique challenges associated with studying ACP2 protein complexes and increase the likelihood of identifying physiologically relevant interactions.

How do ACP2 antibodies contribute to investigating the role of ACP2 in influenza virus replication?

ACP2 antibodies have emerged as essential tools for understanding the critical role of ACP2 in influenza virus replication. Research has established that ACP2 is required for membrane fusion during influenza virus entry, making it a potential target for antiviral development . Several methodological approaches utilizing ACP2 antibodies have advanced this field:

• Validation of knockdown efficiency: ACP2 antibodies enable precise quantification of knockdown efficiency in siRNA experiments targeting ACP2. Western blot analysis with ACP2 antibodies confirmed that siACP2 effectively reduced endogenous ACP2 protein levels in both non-infected and infected cells, providing a solid foundation for subsequent functional studies .

• Viral protein expression analysis: Following ACP2 knockdown, researchers used antibodies against viral proteins (NP, M1, and M2) alongside ACP2 antibodies to demonstrate that ACP2 depletion significantly reduced expression of these viral components. This approach directly linked ACP2 function to viral replication efficiency .

• Immunofluorescence microscopy: ACP2 antibodies, in combination with viral protein antibodies (e.g., anti-NP), allow visualization of the spatial relationship between ACP2 and viral components during infection. Confocal microscopy revealed that GFP expression after rPR8-GFP virus addition was decreased by up to 90% following ACP2 depletion .

• Cross-strain validation: ACP2 antibodies facilitated studies demonstrating that ACP2 is required for the replication of diverse influenza strains, including:

  • A/California/07/2009 (H1N1)

  • A/Perth/16/2009 (H3N2)

  • B/Florida/04/2006

  • A/EM/Korea/W266/2007 (H7N4)

  • A/EM/Korea/W152/2006 (H7N7)

• Mechanistic investigations: By combining ACP2 antibodies with markers for different endocytic compartments, researchers determined that ACP2 specifically functions in the membrane fusion process during viral entry, rather than affecting other viral life cycle stages .

These applications demonstrate how ACP2 antibodies serve as critical reagents for elucidating the molecular mechanisms underlying influenza virus replication and identifying potential therapeutic targets.

What experimental protocols can demonstrate the specificity of ACP2's role in influenza virus replication versus other viral infections?

Establishing the specificity of ACP2's role in influenza virus replication versus other viral infections requires carefully designed experimental protocols. Research has shown that ACP2 is crucial for influenza virus entry but not for other RNA viruses like Ebola virus and HCV . Here are methodological approaches to demonstrate this specificity:

• Comparative viral infection assays:

  • Deplete ACP2 using validated siRNA approaches (confirmed by Western blot with anti-ACP2 antibodies)

  • Infect cells with influenza viruses and other RNA viruses (e.g., Ebola virus, HCV, VSV)

  • Compare viral replication efficiency using:

    • Virus-specific reporter systems

    • qRT-PCR for viral RNA quantification

    • Immunoblotting for viral proteins

    • Viral titer determination (TCID50 assays)

• Rescue experiments:

  • Knockdown endogenous ACP2

  • Reintroduce wild-type or mutant ACP2 variants using expression constructs resistant to siRNA

  • Assess the ability of different ACP2 variants to restore influenza virus replication

  • Compare rescue efficiency between influenza and other viruses

• Time-of-addition experiments:

  • Add ACP2 inhibitors (pharmacological or antibody-based) at different time points during viral infection

  • Determine the temporal window during which ACP2 inhibition affects influenza replication

  • Compare with timing effects on other viral infections to identify stage-specific requirements

• Subcellular localization studies:

  • Use immunofluorescence with anti-ACP2 antibodies to track ACP2 localization during infection with different viruses

  • Co-stain with markers for viral components and cellular compartments

  • Analyze colocalization patterns to identify virus-specific interactions with ACP2

• Biochemical interaction assays:

  • Perform co-immunoprecipitation with anti-ACP2 antibodies during infection with different viruses

  • Identify virus-specific protein interactions through mass spectrometry

  • Validate interactions through reciprocal co-IP and pulldown assays

These protocols provide complementary evidence for the specificity of ACP2's role in influenza virus replication. Research has already established that "ACP2 was indeed crucial for influenza virus replication... in contrast to other RNA viruses such as Ebola virus and HCV," highlighting the potential of ACP2 as a target for influenza-specific therapeutics .

What are the optimal protocols for Western blot analysis using ACP2 antibodies?

Successful Western blot analysis with ACP2 antibodies requires attention to several critical parameters. Based on validated experimental conditions, the following protocol is recommended:

Sample Preparation:

• Extract proteins from cells or tissues using appropriate lysis buffers containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare 30 μg of protein per sample under reducing conditions

Gel Electrophoresis:

• Use 5-20% gradient SDS-PAGE gels for optimal resolution of ACP2

• Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Protein Transfer:

• Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes

• Verify transfer efficiency with Ponceau S staining

Blocking and Antibody Incubation:

• Block membrane with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Incubate with primary anti-ACP2 antibody at the validated concentration:

  • For Boster Bio A06554-1 and A06554-2: 0.5 μg/mL overnight at 4°C

  • For Proteintech 15236-1-AP: 1:500-1:1000 dilution

• Wash with TBS-0.1% Tween (3 times, 5 minutes each)

• Incubate with appropriate secondary antibody (e.g., goat anti-rabbit IgG-HRP) at 1:5000 dilution for 1.5 hours at room temperature

• Wash with TBS-0.1% Tween (3 times, 5 minutes each)

Detection:

• Develop using an enhanced chemiluminescent (ECL) detection system

• Expose to X-ray film or capture images using a digital imaging system

• Expect to observe ACP2 bands at:

  • 52 kDa in human cell lines and neural tissues

  • 76 kDa in liver and pancreatic tissues

  • 63 kDa and 52 kDa in kidney and certain cell lines

Controls and Validation:

• Include positive control samples with known ACP2 expression:

  • Human: Hela, U251, MCF-7, Caco-2, 293T, A549, SH-SY5Y cell lysates

  • Mouse: Brain, liver, pancreas, kidney tissues

  • Rat: Brain, liver, pancreas tissues

• Consider including ACP2 knockdown samples as negative controls

Following this protocol will help ensure consistent and reliable detection of ACP2 in Western blot applications.

How should researchers optimize immunofluorescence protocols for ACP2 detection in different cell types?

Optimizing immunofluorescence (IF) protocols for ACP2 detection requires careful consideration of ACP2's lysosomal localization and expression patterns across different cell types. Based on available data, the following optimized protocol is recommended:

Cell Preparation:

• Culture cells on glass coverslips or chamber slides to 70-80% confluence

• Consider cell type-specific considerations:

  • HepG2 cells have been validated for ACP2 IF detection

  • For other cell types, verify expression levels via Western blot before attempting IF

Fixation and Permeabilization:

• Fixation options:

  • 4% paraformaldehyde (PFA) for 15 minutes at room temperature (preserves morphology)

  • Methanol for 10 minutes at -20°C (may provide better access to some lysosomal epitopes)

• Permeabilization:

  • For PFA-fixed cells: 0.1-0.2% Triton X-100 for 10 minutes

  • For lysosomal proteins like ACP2, saponin (0.1%) may provide gentler permeabilization while preserving lysosomal integrity

Blocking and Antibody Incubation:

• Block with 5% normal serum (from the same species as the secondary antibody) in PBS for 1 hour

• Incubate with primary anti-ACP2 antibody:

  • For Proteintech 15236-1-AP: Use at 1:10-1:100 dilution

  • Test multiple dilutions to determine optimal signal-to-noise ratio

  • Incubate overnight at 4°C in a humidified chamber

• Wash 3 times with PBS, 5 minutes each

• Incubate with fluorophore-conjugated secondary antibody at manufacturer's recommended dilution for 1 hour at room temperature

• Wash 3 times with PBS, 5 minutes each

Co-staining Considerations:

• For confirming lysosomal localization, co-stain with established lysosomal markers:

  • LAMP1 or LAMP2 antibodies

  • LysoTracker dye (add to live cells before fixation)

• For viral infection studies, co-stain with viral protein antibodies (e.g., anti-NP for influenza)

Counterstaining and Mounting:

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

• Mount using anti-fade mounting medium

• Seal edges with nail polish for long-term storage

Imaging and Analysis:

• Capture images using confocal microscopy for optimal resolution of lysosomal structures

• For quantitative analysis, collect z-stacks to capture the full cellular volume

• Use appropriate controls to set exposure settings and thresholds:

  • Omit primary antibody (secondary antibody only)

  • Use ACP2 knockdown cells as negative controls

Cell Type-Specific Optimization:

• For neuronal cells: Extend permeabilization time to ensure access to complex cellular projections

• For polarized epithelial cells: Image both apical and basal compartments

• For highly autofluorescent cells: Consider adding an autofluorescence quenching step

Following this protocol while incorporating cell type-specific adaptations will maximize the likelihood of successful ACP2 detection in immunofluorescence applications.

What approaches can resolve contradictory results between different ACP2 antibodies in research applications?

• Epitope mapping comparison:

  • Determine the epitope regions recognized by each antibody

  • Assess whether epitopes might be differentially affected by:

    • Post-translational modifications

    • Protein conformational changes

    • Protein-protein interactions

    • Alternative splicing variants

• Cross-validation with orthogonal techniques:

  • Complement antibody-based detection with non-antibody methods:

    • Mass spectrometry to verify protein identity

    • RNA-seq or qRT-PCR to correlate protein detection with transcript levels

    • CRISPR-based tagging of endogenous ACP2 with fluorescent proteins

• Genetic validation approaches:

  • Generate ACP2 knockdown or knockout models to create negative controls

  • Test all antibodies against these genetic controls to verify specificity

  • Research has demonstrated successful ACP2 knockdown verification by Western blot, providing a robust validation strategy

• Methodological optimization:

  • For each antibody, systematically optimize:

    • Antibody concentration and incubation conditions

    • Sample preparation methods (lysis buffers, reducing vs. non-reducing conditions)

    • Blocking reagents to minimize background

    • Detection systems with appropriate sensitivity

• Application-specific considerations:

  • Western blot: Different antibodies may detect distinct bands (52 kDa vs. 76 kDa)

    • Run longer gels to resolve potential isoforms

    • Use gradient gels (5-20%) for optimal resolution

  • Immunofluorescence: Evaluate fixation and permeabilization conditions

    • PFA vs. methanol fixation may expose different epitopes

    • Saponin vs. Triton X-100 permeabilization for lysosomal proteins

• Tissue and cell type specificity analysis:

  • Compare antibody performance across multiple validated cell types:

    • Human: Hela, U251, MCF-7, T-47D, HepG2, HL-60 cells

    • Mouse and rat: Brain, liver, pancreas, kidney tissues

  • Different antibodies may perform optimally in specific cellular contexts

• Vendor communication:

  • Contact antibody manufacturers for technical support

  • Request detailed validation data beyond catalog information

  • Inquire about known limitations or application-specific recommendations

By systematically implementing these approaches, researchers can resolve discrepancies between different ACP2 antibodies and establish reliable protocols for their specific experimental systems.