AHSA2P Antibody

What is AHSA2P and what is its biological function?

AHSA2P functions as a co-chaperone that stimulates HSP90 ATPase activity. It significantly enhances the functionality of the Hsp90 chaperone system, which plays a critical role in maintaining protein homeostasis, particularly under stress conditions such as heat shock or increased metabolic activity . As a co-chaperone, AHSA2P interacts directly with the HSP90 complex to regulate its ATP-dependent chaperone activity, thereby influencing protein folding, stability, and functional regulation of numerous client proteins involved in signal transduction, transcriptional regulation, and cell cycle control.

The protein has several synonyms in the literature, including AHA1, activator of heat shock 90kDa protein ATPase homolog 2, and Hch1 . This nomenclature diversity reflects its evolutionary conservation and functional importance across different model systems.

What species reactivity is available for AHSA2P antibodies?

Current commercially available AHSA2P antibodies demonstrate cross-reactivity with multiple species, most commonly:

• Human

• Mouse

• Rat

This multi-species reactivity is documented across different antibody sources . The conservation of immunogenic epitopes across these species suggests evolutionary preservation of key functional domains within the AHSA2P protein. Researchers should note that while cross-reactivity is reported, the affinity and specificity may vary between species, necessitating validation for each experimental model system.

What are the validated applications for AHSA2P antibodies?

AHSA2P antibodies have been validated for multiple experimental techniques, including:

• Western blotting (WB) - for protein expression quantification

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) - for localization studies

• Immunocytochemistry/Immunofluorescence (ICC/IF) - for subcellular localization

• Enzyme-linked immunosorbent assay (ELISA) - for protein quantification

For Western blotting applications, the predicted molecular weight of AHSA2P is approximately 33 kDa . Validation data confirms detection in multiple tissue lysates including liver, kidney, brain, heart, and skeletal muscle samples .

What are the recommended protocols for optimizing AHSA2P antibody dilutions?

Optimal antibody dilution must be empirically determined for each application. Based on validation data, the following starting dilutions are recommended:

For Western blotting:

• Initial dilution: 1:500

• Sample types successfully tested: Rat liver and kidney lysates; Mouse brain, heart, and skeletal muscle lysates

• Secondary antibody: Goat polyclonal to rabbit IgG at 1:50000 dilution

For Immunohistochemistry (paraffin-embedded tissues):

• Dilution range: 1:100 (for intense staining) to 1:500 (for weaker signal)

• Successfully validated on human gastric cancer tissue samples

• Recommended starting concentration: 1-4 μg/ml

For Immunocytochemistry/Immunofluorescence:

• Recommended starting concentration: 1-4 μg/ml

A titration series is recommended for each new lot of antibody and for each new experimental condition. Sequential dilutions (e.g., 1:100, 1:200, 1:500, 1:1000) should be tested to identify the optimal signal-to-noise ratio for your specific application.

What sample preparation considerations are critical for AHSA2P detection?

Sample preparation significantly impacts AHSA2P detection quality. Key considerations include:

For protein extraction in Western blotting:

• Complete protease inhibitor cocktails should be included in lysis buffers

• Maintain cold temperatures during extraction to prevent protein degradation

• Consider the solubility properties of membrane-associated proteins if investigating AHSA2P interactions with membrane-bound HSP90

For immunohistochemistry:

• Fixation method affects epitope accessibility; paraformaldehyde fixation is commonly used

• Antigen retrieval methods may be necessary due to cross-linking during fixation

• Paraffin-embedded human tissue samples have been successfully used for AHSA2P detection

For all applications, freshly prepared samples generally yield superior results compared to stored samples.

What controls should be included when using AHSA2P antibodies?

Rigorous experimental design requires appropriate controls:

Positive controls:

• Tissues with known AHSA2P expression (e.g., liver, kidney, brain)

• Recombinant AHSA2P protein (particularly useful as the antibody was raised against a recombinant fragment corresponding to amino acids 1-150 of human AHSA2P)

Negative controls:

• Samples from AHSA2P knockout models (if available)

• Primary antibody omission control

• Isotype control (rabbit IgG at equivalent concentration)

• Pre-absorption of antibody with immunizing peptide

Loading controls for Western blot:

• Housekeeping proteins (e.g., GAPDH, β-actin)

• Total protein staining methods (e.g., Ponceau S)

How can researchers validate antibody specificity for AHSA2P?

Antibody validation is critical for research reproducibility. Multiple approaches should be used:

Genetic validation:

• AHSA2P knockdown using siRNA or shRNA

• AHSA2P knockout models

• Overexpression of tagged AHSA2P protein

Biochemical validation:

• Western blot should show a single band at the predicted molecular weight (33 kDa)

• Pre-absorption test with immunizing peptide should abolish signal

• Multiple antibodies targeting different AHSA2P epitopes should show similar patterns

Orthogonal validation:

• Correlation between protein detection (antibody-based) and mRNA expression (PCR-based)

• Mass spectrometry confirmation of immunoprecipitated proteins

Some commercial AHSA2P antibodies have undergone specificity verification on protein arrays containing the target protein plus 383 other non-specific proteins , providing additional confidence in their specificity.

What is known about AHSA2P expression patterns across different tissues?

While comprehensive expression atlases for AHSA2P are still developing, experimental evidence suggests:

• Expression in multiple tissues including liver, kidney, brain, heart, and skeletal muscle

• Detectable expression in gastric cancer tissue samples by IHC

When designing experiments, researchers should consider:

• Basal expression levels may vary significantly between tissues

• Expression may be induced under stress conditions, given AHSA2P's role in the heat shock response

• Control samples from the same tissue type are essential for comparative studies

How does AHSA2P interact with the HSP90 chaperone system?

AHSA2P functions as a co-chaperone that stimulates HSP90 ATPase activity . The interaction involves:

• Direct binding to HSP90 to modulate its conformational states

• Enhancement of ATP hydrolysis, which is critical for the chaperone cycle

• Potentially distinct roles under different cellular stress conditions

For experimentally investigating these interactions, researchers might consider:

• Co-immunoprecipitation studies using AHSA2P antibodies

• Proximity ligation assays to visualize in situ interactions

• Activity assays measuring HSP90 ATPase activity in the presence/absence of AHSA2P

Understanding the stoichiometry and dynamics of these interactions requires careful experimental design with appropriate controls.

What are common issues when using AHSA2P antibodies and how can they be resolved?

High background in immunohistochemistry:

• Increase blocking time or blocker concentration

• Optimize primary antibody dilution (try 1:200-1:500)

• Ensure thorough washing between steps

• Use species-specific blocking reagents to minimize non-specific binding

Weak or no signal in Western blot:

• Verify protein transfer efficiency

• Increase protein loading (start with 20-50 μg total protein)

• Reduce antibody dilution (try 1:250 instead of 1:500)

• Optimize exposure time

• Consider alternative extraction methods if AHSA2P is in insoluble fractions

Multiple bands in Western blot:

• Verify sample integrity (fresh preparation, complete protease inhibition)

• Increase washing stringency

• Consider post-translational modifications or isoforms

• Compare with predicted molecular weight (33 kDa)

Low reproducibility:

• Standardize protocols for sample preparation

• Use consistent antibody lots when possible

• Document all experimental conditions thoroughly

How should researchers optimize fixation methods for AHSA2P detection in immunohistochemistry?

Fixation significantly impacts epitope accessibility and antibody binding efficiency:

Paraformaldehyde fixation:

• 4% paraformaldehyde in PBS, pH 7.4

• Fixation time: 24-48 hours for tissues, 10-15 minutes for cells

• Compatible with most AHSA2P antibodies

Antigen retrieval methods:

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

• Enzymatic retrieval using proteinase K may be considered for heavily fixed samples

• Optimization of retrieval time is necessary (typically 10-20 minutes)

Commercial AHSA2P antibodies have been successfully used for IHC on paraffin-embedded human gastric cancer tissue at dilutions of 1:100 , suggesting compatibility with standard formalin fixation and paraffin embedding protocols.

What considerations are important when selecting between polyclonal and monoclonal AHSA2P antibodies?

The search results indicate available AHSA2P antibodies are primarily rabbit polyclonal antibodies . When selecting antibodies:

Polyclonal antibodies (currently available):

• Advantages: Multiple epitope recognition, robust signal amplification, tolerance to minor protein denaturation

• Limitations: Potential batch-to-batch variation, higher background in some applications

• Current applications: Validated for WB, IHC-P, ICC/IF, and ELISA

Monoclonal antibodies (if developed in future):

• Would offer: Higher reproducibility, potentially greater specificity

• Would be optimal for: Distinguishing between closely related proteins, quantitative applications

Given the current market availability, researchers working with AHSA2P should implement rigorous validation for each new lot of polyclonal antibody to ensure consistent experimental results.

How might AHSA2P antibodies be utilized in studying stress response mechanisms?

Given AHSA2P's role in enhancing HSP90 chaperone function during stress conditions , antibodies against this protein enable several research avenues:

Stress response kinetics:

• Time-course experiments tracking AHSA2P expression following heat shock, oxidative stress, or other cellular stressors

• Co-localization studies with HSP90 and client proteins under various stress conditions

• Analysis of AHSA2P post-translational modifications in response to stress

Tissue-specific stress responses:

• Comparative analysis of AHSA2P expression and localization across tissues during systemic stress

• Investigation of cell-type specific roles in heterogeneous tissues

Methodological approaches might include:

• Live-cell imaging with fluorescently tagged AHSA2P antibodies (for cell-permeable variants)

• Chromatin immunoprecipitation studies to investigate potential transcriptional regulatory roles

• High-content screening approaches to identify modulators of AHSA2P-HSP90 interactions

What technical innovations might improve AHSA2P antibody applications?

Several emerging technologies could enhance AHSA2P research:

Next-generation antibody formats:

• Single-domain antibodies with improved tissue penetration

• Recombinant antibody fragments with defined binding characteristics

• Bispecific antibodies targeting AHSA2P and interacting partners simultaneously

Advanced detection methods:

• Super-resolution microscopy for precise subcellular localization

• Multiplexed imaging to simultaneously visualize multiple components of the chaperone system

• Proximity-dependent labeling to identify novel AHSA2P interacting partners

These approaches would extend beyond current validated applications (WB, IHC-P, ICC/IF, ELISA) to provide dynamic, systems-level insights into AHSA2P function.