HSPA1A Antibody, HRP conjugated

What is HSPA1A and why is it an important research target?

HSPA1A is a member of the heat shock protein 70 (HSP70) family that plays crucial roles in protein homeostasis. In cooperation with other chaperones, HSPA1A stabilizes preexistent proteins against aggregation and mediates the folding of newly translated polypeptides in the cytosol and within organelles. These chaperones function by recognizing nonnative conformations of other proteins, binding to extended peptide segments with hydrophobic character that are exposed during translation, membrane translocation, or following stress-induced damage . Additionally, HSPA1A has been identified as serving as a post-attachment receptor for rotavirus A, facilitating viral entry into cells . The multifunctional nature of HSPA1A makes it an important target for research in fields ranging from protein folding mechanisms to understanding viral pathogenesis.

What are the key specifications of commercially available HRP-conjugated HSPA1A antibodies?

Commercially available HRP-conjugated HSPA1A antibodies typically have the following specifications:

• Host: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Species Reactivity: Human, Mouse, Rat

• Conjugation: HRP (Horseradish Peroxidase)

• Recommended Dilutions:

  • Western Blot: 1:100-1000

  • IHC-P (Immunohistochemistry-Paraffin): 1:100-500

These antibodies are designed to recognize HSPA1A from multiple species, making them versatile tools for comparative studies across human and rodent models.

How does HSPA1A function in antigen presentation?

HSPA1A functions as a molecular chaperone that has been suggested to shuttle human leukocyte antigen (HLA) epitope precursors from the proteasome to the transporter associated with antigen processing . Research has demonstrated that peptides chaperoned by HSPA can serve as an effective source of antigens for cross-priming .

Studies have identified 44 different peptides co-purified with HSPA, and the affinity of these peptides to HSPA1A was confirmed using peptide array technology . Four of the HSPA-associated peptides matched with 13 previously reported HLA epitopes, including nine HLA class I and four HLA class II epitopes . This evidence supports HSPA1A's involvement in both HLA class I and class II antigen presentation processes, highlighting its important role in immune system function.

What is the optimal protocol for detecting HSPA1A using HRP-conjugated antibodies in Western blotting?

For optimal detection of HSPA1A using HRP-conjugated antibodies in Western blotting:

• Sample Preparation: Prepare cell or tissue lysates under reducing conditions.

• Electrophoresis: Separate proteins using SDS-PAGE. HSPA1A typically appears at approximately 66 kDa .

• Transfer: Transfer proteins to a membrane (PVDF or nitrocellulose).

• Blocking: Block the membrane with 3% skimmed milk powder in TBS (150 mM NaCl, 10 mM Tris-HCl, pH 7.6) for 3 hours at room temperature .

• Antibody Incubation: Dilute HRP-conjugated anti-HSPA1A antibody 1:100-1000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash the membrane three times for 10 minutes each with TBS containing 0.2% Tween 20 .

• Detection: Apply ECL Plus detection reagents and visualize using a luminescent scanner .

For higher sensitivity and specificity, optimization of antibody concentration and incubation conditions may be required based on your specific experimental setup.

How can HSPA1A antibodies be used for immunohistochemistry in research models?

For immunohistochemical detection of HSPA1A in research models:

• Tissue Preparation: Fix tissue samples and embed in paraffin or prepare frozen sections.

• Deparaffinization and Rehydration: For paraffin sections, deparaffinize with xylene and rehydrate through graded alcohols.

• Antigen Retrieval: Perform heat-induced epitope retrieval (specific buffer may need optimization).

• Blocking: Block endogenous peroxidase activity and non-specific binding.

• Primary Antibody: Apply HRP-conjugated anti-HSPA1A antibody at a dilution of 1:100-500 .

• Washing: Wash thoroughly between steps.

• Substrate Development: Apply a chromogenic substrate for HRP (such as DAB).

• Counterstaining: Counterstain with hematoxylin, dehydrate, and mount.

Research has demonstrated the effectiveness of this approach. For example, IHC has been successfully used to detect elevated HSP70 levels in mouse skin following radiofrequency (RF) treatment, as shown in representative IHC images where HSP70 expression was visualized in RF-treated versus non-treated skin at 24 hours post-treatment .

What are the relative binding affinities of HSPA1A to different peptides in experimental settings?

Research has identified various peptides with different binding affinities to HSPA1A. A comprehensive peptide affinity assay revealed that HSPA1A binds to a specific repertoire of peptides with varying affinities. Below is a selection from a research study showing relative binding affinities:

Where: ++ indicates high relative affinity, + indicates moderate affinity, and 0 indicates no binding .

This data demonstrates that HSPA1A has preferential binding to specific peptide sequences, which is important for understanding its chaperoning functions in antigen presentation and protein folding.

How can HRP-conjugated HSPA1A antibodies be utilized in Simple Western assays?

Simple Western assays represent an advanced application for HRP-conjugated HSPA1A antibodies. Research has demonstrated effective protocols:

• Sample Preparation: Prepare cell lysates (e.g., Jurkat human acute T cell leukemia cell line) at 0.2 mg/mL concentration.

• Control Preparation: Include both untreated cells and cells subjected to heat shock to demonstrate dynamic changes in HSPA1A expression.

• Antibody Dilution: Use the anti-HSPA1A antibody at approximately 0.5 μg/mL.

• Detection System: Run the assay under reducing conditions using a 12-230 kDa separation system.

• Expected Results: A specific band for HSPA1A/HSP70 should be detected at approximately 66 kDa .

This technique has been successfully demonstrated in research comparing untreated versus heat-shocked Jurkat cells, and in comparing expression across different cell lines such as Jurkat human cells and C2C12 mouse myoblast cells .

What is the role of HSPA1A in comparative cross-species immunological research?

HSPA1A is highly conserved across species, making it valuable for comparative immunological research. The HRP-conjugated HSPA1A antibodies that demonstrate reactivity with human, mouse, and rat antigens enable researchers to conduct cross-species studies with a single antibody.

Research has shown that HSPA binding specificity is at least partially conserved between cell lines and across species. Studies have identified pairs of peptides with overlapping sequences isolated from different cell lines, as well as peptides derived from different proteins showing high sequence homology . Additionally, some peptides identified in human studies share common sequences with peptides reported as co-purified with mouse HSPA .

This conservation allows researchers to:

• Compare heat shock responses across species

• Validate experimental findings across multiple model organisms

• Study evolutionarily conserved immune mechanisms

• Develop translational research from animal models to human applications

How does HSPA1A participate in immune activation pathways?

Advanced research has elucidated HSPA1A's role in immune activation pathways. Studies using radiofrequency (RF) treatment in mouse models have demonstrated that:

• RF treatment increases HSP70 levels in skin, as detected by Western blotting at 6 and 24 hours post-treatment .

• This increase in HSP70 activates the MyD88 (Myeloid differentiation primary response 88) pathway .

• When comparing wild-type (WT) and MyD88 knockout (KO) mice exposed to RF treatment followed by intradermal OVA (ovalbumin) injection, significant differences in anti-OVA antibody titers were observed .

• The use of MyD88 inhibitory peptides (Pepinh-MyD) before RF treatment and OVA immunization resulted in reduced antibody responses compared to control peptides .

These findings suggest that HSPA1A not only functions as a molecular chaperone but also plays a crucial role in activating innate immune responses through the MyD88 pathway, which subsequently influences adaptive immunity.

How can researchers optimize peptide binding assays for HSPA1A studies?

For optimal peptide binding assays with HSPA1A:

• Peptide Length Considerations: Due to synthesis method restrictions, peptides longer than 20 residues should be truncated to 20-residue fragments with at least 15 residues of overlapping fragments .

• Peptide Synthesis: Synthesize peptides at approximately 5 nmol per spot with acetylated N-terminal ends and C-terminal covalent binding to membranes via polyethylene glycol linkers .

• Blocking Protocol: Block membranes with 3% skimmed milk powder in TBS (150 mM NaCl, 10 mM Tris-HCl, pH 7.6) for 3 hours .

• Protein Concentration: Use 100nM HSPA1A in TBS and incubate overnight at 4°C .

• Detection Method: Use specific anti-HSPA primary antibodies (e.g., 1:1,000 dilution) and HRP-conjugated secondary antibodies (e.g., 1:5,000 dilution) with 1-hour incubations for each .

• Washing Steps: Perform three 10-minute washes with TBS containing 0.2% Tween 20 after each incubation step .

• Visualization: Use ECL Plus detection reagents and quantify signal intensity with a calibrated trans-luminescent scanner .

• Affinity Assessment: Classify peptides as non-binding (intensity lower than double the average value of negative control spots), moderate affinity (intensity higher than twofold increase over corresponding control), or high affinity (intensity higher than fourfold increase over corresponding control) .

This methodological approach has been validated in research identifying HSPA-associated peptides that match with HLA epitopes.

What controls should be included when using HRP-conjugated HSPA1A antibodies in experimental designs?

Robust experimental design with HRP-conjugated HSPA1A antibodies should include the following controls:

• Negative Controls:

  • Antibody-only controls (primary and secondary antibodies without sample) to assess non-specific binding

  • Isotype controls to evaluate background from the antibody class

  • Untreated cell/tissue samples to establish baseline expression levels

• Positive Controls:

  • Heat-shocked cell samples (known to upregulate HSPA1A)

  • Recombinant HSPA1A protein at known concentrations

  • Well-characterized cell lines with documented HSPA1A expression (e.g., Jurkat cells)

• Specificity Controls:

  • Peptide competition assays to confirm antibody specificity

  • Comparing multiple antibodies against the same target

  • Testing across multiple species when using antibodies with cross-reactivity claims

• Technical Controls:

  • Loading controls (e.g., GAPDH) for Western blotting

  • Dilution series to establish the linear range of detection

  • Internal calibration standards for quantitative analyses

Including these controls helps validate results, troubleshoot technical issues, and ensure scientific rigor in HSPA1A research.

How can researchers distinguish between constitutive and inducible forms of HSP70 in experimental data?

Distinguishing between constitutive HSP70 (HSPA8/Hsc70) and inducible HSP70 (HSPA1A) is critical for accurate data interpretation:

• Antibody Selection: Use antibodies specifically raised against unique epitopes of HSPA1A that do not cross-react with HSPA8. The epitope specificity should be verified by the manufacturer or through validation experiments .

• Expression Pattern Analysis:

  • HSPA1A (inducible form) shows low expression under normal conditions but is significantly upregulated following stress (e.g., heat shock, oxidative stress)

  • HSPA8 (constitutive form) shows relatively stable expression under various conditions

  • Temporal analysis can reveal different induction kinetics

• Comparative Blotting: Run parallel Western blots with antibodies specific to HSPA1A and HSPA8 on the same samples. Research has demonstrated this approach by analyzing HSP70 (HSPA1A), HSc70 (HSPA8), and HSP90 levels by Western blotting at 6 and 24 hours after radiofrequency treatment in mouse models .

• Molecular Weight Differentiation: Although similar, slight differences in molecular weight can sometimes be distinguished in high-resolution gel systems. Both forms typically appear around 66-70 kDa .

• Functional Assays: Some studies use functional differences between the two forms to distinguish them, such as ATP-binding capacity or interaction with specific co-chaperones.

By employing these strategies, researchers can accurately differentiate between constitutive and inducible forms of HSP70, leading to more precise interpretation of experimental results.