APPL1 Antibody

What applications are APPL1 antibodies validated for?

APPL1 antibodies have been extensively validated for multiple research applications. According to available data, APPL1 antibodies demonstrate consistent performance across several techniques:

Most commercially available APPL1 antibodies require optimization in each experimental system to obtain optimal results. While the table provides general dilution guidelines, it's recommended to perform titration experiments for your specific application and sample type .

What species reactivity do APPL1 antibodies demonstrate?

Most commercially available APPL1 antibodies show cross-reactivity with human, mouse, and rat APPL1 proteins. Some antibodies also demonstrate reactivity with monkey (Mk) samples . For specific applications:

• Positive Western blot detection has been demonstrated in human tissues (brain, heart), mouse tissues (brain, liver, ovary), and multiple cell lines including HEK-293, HeLa, HT-1080, and C2C12 cells .

• Immunoprecipitation has been validated primarily in mouse brain tissue .

• Immunohistochemistry works effectively with human breast cancer and ovary tumor tissues .

Some antibodies may have predicted reactivity with additional species (pig, bovine, horse, sheep, rabbit, dog, chicken, and Xenopus), though these require experimental validation by the researcher .

What is the molecular weight of APPL1 detected by antibodies?

APPL1 antibodies typically detect a protein band at approximately 80-82 kDa. The calculated molecular weight based on amino acid sequence is 80 kDa (709 amino acids), which aligns with the observed molecular weight in experimental systems . This consistency between calculated and observed molecular weights provides confidence in antibody specificity.

What are the optimal storage conditions for APPL1 antibodies?

Most APPL1 antibodies are supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . The recommended storage conditions are:

• Store at -20°C

• Antibodies are generally stable for one year after shipment when stored properly

• Aliquoting is typically unnecessary for -20°C storage

• Some preparations (20μl sizes) may contain 0.1% BSA

These storage conditions maintain antibody integrity and activity. Repeated freeze-thaw cycles should be avoided to prevent antibody degradation and loss of binding capacity .

What controls should be included when working with APPL1 antibodies?

Based on published research methodologies, the following controls are recommended:

• Positive controls: Use tissues or cell lines with known APPL1 expression such as human brain tissue, HEK-293 cells, HeLa cells, or mouse brain tissue .

• Negative controls:

  • Primary antibody omission

  • Non-specific IgG from the same host species (e.g., rabbit IgG-agarose for rabbit APPL1 antibodies)

  • siRNA-mediated silencing of APPL1 as a specificity control (95% silencing efficiency has been achieved in published studies)

• For co-localization studies: Include appropriate markers for subcellular compartments (e.g., α-tubulin, GM130, TGN38, caveolin-1, BiP/Grp78, RAB5, actin) .

How can I validate the specificity of APPL1 antibodies?

Several approaches have been used in published research to validate APPL1 antibody specificity:

• siRNA knockdown: Transfect cells with APPL1-specific siRNA and confirm reduction in signal by Western blot and immunofluorescence. Published studies have achieved 95% knockdown efficiency with undetectable levels of APPL1 protein by immunoblot analysis (using 20 μg total protein) .

• Immunoblot analysis: Verify single band at the expected molecular weight (80 kDa) in tissues known to express APPL1 .

• Cross-reactivity testing: Confirm the antibody doesn't cross-react with related proteins (e.g., APPL2). Published studies have demonstrated that anti-APPL1 and anti-APPL2 antibodies are highly specific and do not cross-react .

• Immunoprecipitation followed by mass spectrometry: Use the "top ten" approach for immunoprecipitated APPL1 from human skeletal muscle to verify antibody accuracy .

How can APPL1 antibodies be used to study protein-protein interactions?

APPL1 antibodies have been successfully employed to study various protein-protein interactions through several techniques:

• Co-immunoprecipitation: APPL1 antibodies have been used to investigate interactions between APPL1 and other proteins. For example, studies have demonstrated that APPL1-YFP fusion proteins co-immunoprecipitate with endogenous APPL1, suggesting homotypic interactions . The protocol typically involves:

  • Using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Inclusion of appropriate negative controls (e.g., mouse IgG-agarose)

  • Western blot analysis of immunoprecipitates for interacting proteins

• Yeast two-hybrid analysis: Complementary to co-IP, this approach has been used to map specific domain interactions, demonstrating that the BAR domain is necessary and sufficient for APPL-APPL interactions .

• Co-localization studies: Immunofluorescence with APPL1 antibodies combined with markers for subcellular compartments has been used to investigate spatial associations of APPL1 with potential interacting proteins .

It's important to note that while strong co-immunoprecipitation of APPL1 and ADIPORs has been reported in cell-based systems, this interaction can be difficult to observe in human skeletal muscle, likely due to the transient or weak nature of the interaction .

What methodological considerations are important when studying APPL1's role in Akt signaling?

APPL1 has been shown to regulate Akt signaling, and several methodological approaches can be used to investigate this relationship:

• Quantification of active Akt: Use phosphorylation-specific antibodies against phospho-Thr-308-Akt to detect active Akt. Studies have shown that expression of GFP-APPL1 reduced the level of active Akt by approximately twofold compared to control cells expressing GFP .

• TIRF microscopy: This technique has been used to visualize and quantify the levels of active Akt in paxillin-containing adhesions. Studies demonstrated that the amount of active Akt in adhesions in APPL1-expressing cells was decreased 1.7-fold compared to control cells .

• Genetic manipulation: Compare active Akt levels between:

  • Cells overexpressing GFP-APPL1

  • Cells with APPL1 knockdown using siRNA

  • Cells expressing APPL1 mutants (e.g., GFP-APPL1-ΔPTB that doesn't affect active Akt levels)

• Controls and normalization: When quantifying fluorescence intensity of active Akt, use appropriate software (e.g., MetaMorph) and normalize to control samples .

How can I troubleshoot non-specific background when using APPL1 antibodies in immunostaining?

When encountering background issues with APPL1 antibodies in immunohistochemistry or immunofluorescence, consider these evidence-based troubleshooting approaches:

• Antigen retrieval optimization: Published protocols suggest using TE buffer pH 9.0 for antigen retrieval, with citrate buffer pH 6.0 as an alternative .

• Antibody titration: The recommended dilution range for immunohistochemistry is 1:20-1:200, but optimal concentration should be determined empirically for each application and tissue type .

• Blocking optimization: Increase the blocking time or concentration of blocking agent (typically BSA or serum from the same species as the secondary antibody).

• Secondary antibody controls: Include controls omitting the primary antibody to identify non-specific binding of the secondary antibody.

• Fixation method consideration: Different fixation methods can affect epitope accessibility and antibody performance.

How does APPL1 expression vary across different tissue types?

APPL1 shows differential expression across tissue types. Based on antibody validation studies:

• Strongly positive in:

  • Brain tissue (human and mouse)

  • Heart tissue (human)

  • Liver tissue (mouse)

  • Ovary tissue (mouse)

  • Breast cancer tissue (human)

• Cell line expression:

  • HEK-293 cells

  • HeLa cells

  • HT-1080 cells

  • C2C12 cells

  • HepG2 cells

Studies examining APPL1 expression in insulin-resistant states found that APPL1 protein levels were significantly increased in skeletal muscle from type 2 diabetic participants (2.42 ± 0.25 arbitrary units) compared with lean control (1.78 ± 0.18) and obese control participants (1.74 ± 0.12; p < 0.05) . This suggests APPL1 upregulation may represent a compensatory mechanism in insulin-resistant states.

How can APPL1 antibodies be used to investigate subcellular localization?

APPL1 antibodies have been extensively used to study the protein's subcellular localization through immunofluorescence. Key methodological considerations include:

• Dilution range: For immunofluorescence/ICC, the recommended dilution is 1:50-1:500 or 1:100-1:400 .

• Detection of membrane-associated APPL1: Studies have shown that full-length APPL-YFP fusion proteins associate with cytosolic membrane structures that undergo movement, fusion, and fission events. Live cell imaging is particularly valuable for observing these dynamic processes .

• Domain-specific localization: Research has demonstrated that:

  • BAR domains localize to tubular membrane structures

  • PH domains localize to the plasma membrane, cytosolic vesicles, and distinct nuclear/perinuclear structures

  • PTB domains localize to cytosolic membrane structures

• Co-localization studies: For comprehensive analysis, pair APPL1 staining with markers for different subcellular compartments including α-tubulin, GM130, TGN38, caveolin-1, BiP/Grp78, RAB5, and actin .

What methods are effective for quantifying changes in APPL1 expression levels?

For accurate quantification of APPL1 expression changes, researchers have employed several complementary approaches:

• Western blotting quantification:

  • Recommended dilution range: 1:1000-1:10000 or 1:2000

  • Loading control: β-actin is commonly used

  • Densitometric analysis: Use appropriate software to quantify band intensity

  • Data presentation: Express as fold change relative to control or arbitrary units

• qRT-PCR for mRNA expression:

  • Studies have shown that APPL1 mRNA increases significantly in obese control and type 2 diabetic participants compared to lean controls

  • This can be correlated with protein level changes to understand transcriptional regulation

• Immunofluorescence quantification:

  • Software-based measurement of fluorescence intensity

  • Studies have used MetaMorph software to quantify levels of APPL1 in individual cells

  • Data can be expressed as percentage of control or arbitrary fluorescence units

When comparing expression levels across experimental conditions, it's critical to maintain consistent imaging parameters, exposure times, and quantification methods to ensure reliable results.

How can APPL1 antibodies be used to study dynamic membrane targeting?

APPL1 functions as a dynamic scaffold that modulates RAB5-associated signaling endosomal membranes. Advanced applications for studying this include:

• Live cell imaging: APPL-YFP fusion proteins can be monitored in real-time to observe membrane structures undergoing movement, fusion, and fission events .

• Domain-specific membrane targeting: Studies have revealed that:

  • All three domains (minimal BAR, PH, and PTB) can target to cell membranes when overexpressed

  • The BAR domain is necessary and sufficient for APPL-APPL interactions

  • PH and PTB domains are sufficient for in vitro phosphoinositide binding

• RAB5 recruitment analysis: Overexpression of full-length APPL-YFP fusion proteins is sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, providing a methodological approach to study this interaction .

• FRAP (Fluorescence Recovery After Photobleaching): This technique can be used to measure the dynamics of APPL1 association with membranes.

What are the methodological considerations for studying APPL1's role in adiponectin signaling?

APPL1 plays an important role in adiponectin signaling, and several methodological approaches can be employed to investigate this function:

• Co-immunoprecipitation limitations: It's important to note that while co-immunoprecipitation of APPL1 and adiponectin receptors (ADIPORs) has been reported in cell-based systems, this interaction can be difficult to detect in human skeletal muscle. This may be because the interaction is too weak to be maintained during the immunoprecipitation procedure .

• Comparative expression analysis: Studies have shown that in insulin-resistant states:

  • APPL1 mRNA and protein levels are increased in type 2 diabetic skeletal muscle

  • ADIPOR1 mRNA expression is significantly increased in obese compared with lean controls

  • These changes may represent compensatory mechanisms in response to decreased adiponectin levels

• Combined protein and mRNA analysis: For comprehensive understanding, researchers should measure both APPL1 protein levels (by Western blotting) and mRNA expression (by qRT-PCR) to distinguish between transcriptional and post-transcriptional regulation.

• Tissue-specific considerations: Expression patterns and interactions may differ between tissues and cell types, necessitating validation in the specific system under investigation.

What experimental approaches can resolve contradictory findings about APPL1 function?

When addressing contradictory findings regarding APPL1 function, researchers should consider these methodological approaches:

• Domain-specific mutant analysis: Studies have demonstrated that different APPL1 domains contribute to distinct functions:

  • The GFP-APPL1-ΔPTB mutant does not affect active Akt levels, while wild-type APPL1 reduces active Akt

  • The GFP-APPL1-AAA mutant (which alters endosomal localization) does not affect active Akt levels

• Cell type and context dependence: APPL1 function may differ between:

  • Different cell lines (e.g., HT1080 vs. HEK-293)

  • Different tissues (e.g., muscle vs. adipose)

  • Different physiological states (e.g., insulin-sensitive vs. insulin-resistant)

• Technical considerations:

  • Antibody selection: Different antibodies may recognize different epitopes or isoforms

  • Experimental conditions: Cell confluence, serum starvation, and stimulation protocols can impact results

  • Expression levels: Overexpression vs. endogenous protein studies may yield different outcomes

• Integrated multi-omics approach: Combine:

  • Protein-level analysis (Western blot, immunoprecipitation)

  • Transcriptomic analysis (qRT-PCR)

  • Functional assays (e.g., cell migration, Akt activity)

  • Structural studies (domain interactions)