HSPA4 Antibody, FITC conjugated

What is HSPA4 and what are its primary cellular functions?

HSPA4 (Heat Shock 70kDa Protein 4) is a member of the heat shock protein 70 family with a calculated molecular weight of 94 kDa, though the observed molecular weight is typically around 110 kDa . This protein participates in several cellular processes including chaperone-mediated protein complex assembly, protein import into mitochondrial outer membrane, and response to unfolded protein stress . HSPA4 is primarily localized in the cytosol and extracellular exosomes, and possesses ATP binding capabilities that facilitate its chaperone functions . Recent research has demonstrated that HSPA4 plays significant roles in various cancers, influencing critical cellular processes such as proliferation, migration, and survival .

What are the recommended applications for HSPA4 antibody?

The HSPA4 antibody has been validated for multiple experimental applications:

It is highly recommended to titrate the antibody for each specific testing system to achieve optimal results, as the optimal concentration may be sample-dependent .

What is the reactivity profile of HSPA4 antibody?

The HSPA4 antibody exhibits confirmed reactivity with samples from multiple species including human, mouse, rat, and monkey . Published literature has primarily cited its use with human and mouse samples . For researchers working with other species, preliminary validation is advisable as reactivity may vary between antibody clones. The FITC-conjugated variant specifically has been validated with human samples according to manufacturer specifications .

What are the optimal storage conditions for HSPA4 antibody with FITC conjugation?

FITC-conjugated antibodies, including HSPA4 antibody, should be stored at 4°C to maintain fluorophore stability and prevent photobleaching . The unconjugated form can be stored at -20°C and remains stable for one year after shipment when maintained in appropriate buffer conditions (PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) . For long-term preservation of activity, it is important to minimize freeze-thaw cycles, though aliquoting is generally unnecessary for the specific formulation described .

How can HSPA4 antibody be optimized for immunohistochemical detection in cancer tissues?

For optimal immunohistochemical detection of HSPA4 in cancer tissues, the following protocol has been validated:

• Tissue section deparaffinization followed by antigen retrieval using citric acid buffer

• Primary antibody incubation with anti-HSPA4 at 1:200 dilution

• Secondary antibody incubation with IgG HRP-conjugated antibody at 1:400 dilution

• Detection using DAB (3,3'-diaminobenzidine) with hematoxylin counterstaining

For FITC-conjugated antibodies, modifications to this protocol would include omitting the secondary antibody step and implementing appropriate fluorescence detection methods. Notably, antigen retrieval can be performed with either TE buffer at pH 9.0 or alternatively with citrate buffer at pH 6.0, with the former showing superior results in some tissue types .

The evaluation of immunohistochemical results should follow a systematic approach using a scoring system that accounts for both staining percentage (1: 1-24%, 2: 25-49%, 3: 50-74%, 4: 75-100%) and staining intensity (0: no color, 1: brown, 2: light yellow, 3: dark brown) .

What are the current findings regarding HSPA4 expression in cancer and its prognostic significance?

Recent comprehensive pan-cancer analyses have revealed significant associations between HSPA4 expression and various cancer types:

Interestingly, KIRC presents an exception where increased HSPA4 expression associates with extended survival (P=0.023) .

What methodological approaches can be used to investigate HSPA4's role in cancer progression at the single-cell level?

Single-cell analysis of HSPA4 can be conducted using several complementary approaches:

• CancerSEA Database Analysis: This specialized single-cell sequencing database enables examination of correlations between HSPA4 expression and 14 tumor-related cellular functions across 900 cancer cells . The T-SNE plot visualization can delineate HSPA4 expression patterns in single cells within TCGA samples.

• Flow Cytometry with FITC-conjugated HSPA4 Antibody: For intracellular detection, a recommended protocol includes:

  • Cell fixation and permeabilization

  • Staining with 0.40 μg HSPA4-FITC antibody per 10^6 cells in 100 μl suspension

  • Analysis using appropriate fluorescence channels for FITC detection

• Protein-Protein Interaction (PPI) Network Analysis: The STRING database can be utilized to identify proteins potentially interacting with HSPA4, with a recommended confidence score threshold of 0.7. Network visualization and analysis through Cytoscape with the cytoHubba plugin can identify key modules and hub genes associated with HSPA4 .

How can researchers effectively investigate the immunological significance of HSPA4 in the tumor microenvironment?

To investigate HSPA4's relationship with immune components in the tumor microenvironment, researchers can employ several algorithmic and database approaches:

• Immune Infiltration Analysis: Utilize algorithms including MCPCOUNTER, TIDE, EPIC, XCELL, and TIMER through the TIMER2.0 tool to assess correlations between HSPA4 expression and immune cell infiltration levels in tumor tissues .

• Molecular/Immune Subtype Correlation: The "Subtype" module of the TISIDB database allows examination of potential associations between HSPA4 expression and molecular or immune subtypes in prevalent cancers .

• Single-sample Gene Set Enrichment Analysis (ssGSEA): Employ the R "GSVA" package to elucidate associations between HSPA4 expression and 22 immune-related cell types .

• Gene Set Enrichment Analysis (GSEA): This approach can discern differences in biological pathways between high and low HSPA4 expression groups, with visualization through platforms like the Xiantao Academic platform .

These methodologies collectively provide a comprehensive framework for understanding HSPA4's potential role in modulating immune responses within the tumor microenvironment.

What are common technical challenges when using FITC-conjugated HSPA4 antibody and how can they be addressed?

When working with FITC-conjugated HSPA4 antibody, researchers commonly encounter several technical issues:

• Photobleaching: FITC is susceptible to rapid photobleaching during microscopy.

  • Solution: Minimize exposure to light during sample preparation, employ anti-fade mounting media containing photoprotectants, and use appropriate neutral density filters during imaging to reduce excitation light intensity.

• Autofluorescence: Particularly problematic in tissues with high collagen content or formalin-fixed samples.

  • Solution: Implement autofluorescence quenching steps using agents such as Sudan Black B (0.1-0.3%) or sodium borohydride treatment (1mg/ml for 10 minutes).

• Inadequate Signal Intensity: Particularly in tissues with low HSPA4 expression.

  • Solution: Optimize antibody concentration through titration experiments (starting with the recommended 1:50-1:500 range for IF/ICC) . Signal amplification systems such as tyramide signal amplification can be considered for detecting low-abundance targets.

• Non-specific Binding: Can result in high background fluorescence.

  • Solution: Implement more stringent blocking (5-10% normal serum from the same species as the secondary antibody plus 1% BSA) and include 0.1-0.3% Triton X-100 in blocking and antibody dilution buffers to improve specificity.

How can researchers validate specificity when using HSPA4 antibody in new experimental contexts?

Rigorous validation of HSPA4 antibody specificity in new experimental systems should include:

• Positive and Negative Controls:

  • Positive Controls: Include samples known to express HSPA4 such as C6 cells, HEK-293 cells, mouse testis tissue, NIH/3T3 cells, or brain tissues from mouse or rat which have been confirmed to show positive WB results .

  • Negative Controls: Include primary antibody omission controls and, ideally, HSPA4-knockout or knockdown samples if available.

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (HSPA4 fusion protein Ag15581) before application to samples.

  • Signal elimination or significant reduction confirms specificity.

• Cross-Validation with Multiple Detection Methods:

  • Compare results across different techniques (WB, IHC, IF) to ensure consistent detection of the target at the expected molecular weight (110 kDa) .

• siRNA or CRISPR Knockout Validation:

  • Generate HSPA4 knockdown or knockout samples and verify reduced antibody binding proportional to target reduction.

How can HSPA4 antibody be utilized to investigate its role in apoptosis and cell cycle regulation?

Based on findings that HSPA4 knockdown causes cell cycle arrest in the G2-phase and increases apoptosis , researchers can implement the following methodological approaches:

• Flow Cytometry for Cell Cycle Analysis:

  • Use FITC-conjugated HSPA4 antibody (0.40 μg per 10^6 cells) in combination with propidium iodide staining to simultaneously assess HSPA4 expression and cell cycle distribution.

  • Compare cell populations with varying HSPA4 expression levels to determine correlation with G2 arrest.

• Apoptosis Assessment:

  • Combine HSPA4 antibody staining with annexin V and 7-AAD to correlate HSPA4 expression with early and late apoptotic events.

  • For mechanistic studies, examine the relationship between HSPA4 expression and apoptosis-related proteins based on research showing altered expression of these proteins with HSPA4 silencing .

• Western Blot Analysis of Cell Cycle Regulators:

  • After cell sorting based on HSPA4 expression or in HSPA4-manipulated cells, use Western blotting (with recommended 1:500-1:2000 dilution) to analyze cell cycle markers CCND1 and CDK6, which have been shown to be downregulated with HSPA4 silencing .

• PI3K/Akt Signaling Assessment:

  • Investigate the correlation between HSPA4 expression and PI3K/Akt pathway activation, as HSPA4 silencing has been shown to reduce activation of this signaling axis .

What methodological approaches can be employed to explore HSPA4's potential as a diagnostic biomarker?

To investigate HSPA4's biomarker potential, researchers should consider:

• Tissue Microarray (TMA) Analysis:

  • Implement standardized IHC staining protocols (1:20-1:200 dilution) on TMAs containing multiple cancer types alongside matched normal tissues.

  • Use the established scoring system (percentage and intensity) to quantify HSPA4 expression differences .

• ROC Curve Analysis:

  • Generate ROC curves to evaluate HSPA4's diagnostic performance, particularly for cancer types where AUC values exceeding 0.7 have been reported, including LUSC, LUAD, LIHC, ESCA, KIRC, GBM, CESC, COAD, and BRCA .

• Correlation with Pathological Staging:

  • Analyze HSPA4 expression across different cancer stages, especially in KICH, KIRP, BLCA, KIRC, LUAD, and LIHC where correlations with pathological staging have been observed .

• Multiparameter Flow Cytometry:

  • Develop flow cytometry panels incorporating FITC-conjugated HSPA4 antibody with other cancer biomarkers to improve diagnostic sensitivity and specificity.

How might HSPA4 antibody be employed in therapeutic development and monitoring?

Given HSPA4's potential as a therapeutic target in colorectal cancer and its prognostic significance in multiple cancer types , researchers can explore:

• Target Validation Studies:

  • Use FITC-conjugated HSPA4 antibody in high-content screening to identify compounds that modulate HSPA4 expression or function.

  • Implement flow cytometry with HSPA4 antibody to monitor therapy-induced changes in HSPA4 expression.

• Patient Stratification for Clinical Trials:

  • Develop standardized IHC protocols using HSPA4 antibody to stratify patients based on expression levels, particularly relevant for cancers where HSPA4 has strong prognostic value (ESAD, LIHC, GBM, LUAD, LGG, HNSC, KICH, OSCC, and KIRP) .

• Combination Therapy Approaches:

  • Investigate the effects of combining HSPA4-targeting strategies with PI3K/Akt pathway inhibitors, based on findings that HSPA4 knockdown reduces activation of this signaling axis .

• Circulating Tumor Cell Analysis:

  • Adapt HSPA4 antibody protocols for CTC detection and monitoring, potentially combining with liquid biopsy approaches for longitudinal patient monitoring.

What are the methodological considerations for investigating HSPA4's role in cancer immunology?

To explore the intersection of HSPA4 function and cancer immunology, researchers should consider:

• Multiplex Immunofluorescence:

  • Develop protocols combining FITC-conjugated HSPA4 antibody with markers for various immune cell populations to visualize spatial relationships between HSPA4-expressing cells and immune infiltrates in the tumor microenvironment.

• Immune Cell Co-culture Systems:

  • Establish in vitro co-culture systems with HSPA4-manipulated cancer cells and immune components to assess functional interactions.

  • Monitor effects using flow cytometry with HSPA4-FITC antibody (0.40 μg per 10^6 cells in 100 μl suspension) .

• Bioinformatic Analysis of HSPA4 and Immune Parameters:

  • Implement the comprehensive immune-related analysis pipeline including MCPCOUNTER, TIDE, EPIC, XCELL, and TIMER algorithms through TIMER2.0 .

  • Use ssGSEA with the R "GSVA" package to explore associations between HSPA4 expression and 22 immune-related cell populations .