HSPA1L Antibody, FITC conjugated

What is HSPA1L and what cellular functions does it regulate in normal and pathological conditions?

Research has shown that HSPA1L exerts its functions through two primary mechanisms. First, it forms a triple complex with IGF1Rβ (Insulin-like Growth Factor 1 Receptor β) and integrin αV, leading to activation of IGF1Rβ. This activation intensifies EMT-associated cancer stemness and radiation resistance through downstream AKT/NF-κB or AKT/GSK3β/β-catenin pathways. Second, HSPA1L can translocate to the nucleus where it directly binds to the promoter region of β-catenin, functioning as a transcription activator of this important signaling protein that characterizes cancer stem cells by regulating ALDH1 expression .

What is the significance of using FITC-conjugated antibodies for HSPA1L detection in research?

FITC (Fluorescein Isothiocyanate) conjugation to HSPA1L antibodies provides significant advantages for protein detection and localization studies. While unconjugated antibodies require secondary detection systems, FITC-conjugated antibodies allow direct visualization of the target protein through fluorescence microscopy or flow cytometry.

For HSPA1L research, FITC-conjugated antibodies enable researchers to:

• Directly visualize subcellular localization (cytoplasmic versus nuclear) of HSPA1L

• Perform co-localization studies with other proteins of interest (especially IGF1Rβ and integrin αV)

• Quantify expression levels through flow cytometry

• Track dynamic changes in HSPA1L expression or localization in response to experimental conditions

The excitation maximum of FITC at approximately 495nm and emission maximum around 519nm provides a bright green fluorescence that is compatible with most standard fluorescence detection systems, making it an ideal conjugate for immunofluorescence studies of HSPA1L in cancer stem cell research.

What sample types have been validated for HSPA1L antibody detection?

Based on comprehensive validation studies, HSPA1L antibodies have demonstrated reliable detection across multiple sample types. The reactivity pattern provides researchers with confidence when selecting appropriate experimental models.

When working with FITC-conjugated HSPA1L antibodies, researchers should consider that validation may vary from unconjugated versions. It is recommended to perform preliminary validation experiments with positive controls known to express HSPA1L, such as HepG2 cells or cancer tissues with documented HSPA1L expression .

How does HSPA1L expression correlate with cancer stem cell properties, and what methodologies best capture this relationship?

HSPA1L expression demonstrates a strong positive correlation with cancer stem cell properties, particularly in non-small cell lung cancer. Research has revealed that HSPA1L is significantly upregulated in ALDH1high cells (a recognized cancer stem cell population) compared to ALDH1low cells .

To effectively investigate this relationship, researchers should employ a multi-methodological approach:

• Flow cytometry sorting and analysis: Using the ALDEFLUOR assay to separate ALDH1high and ALDH1low populations, followed by HSPA1L detection using FITC-conjugated antibodies. This allows for quantitative correlation between ALDH1 activity and HSPA1L expression .

• Sphere formation assays: Comparing sphere-forming capacity between cells with different HSPA1L expression levels (wild-type, knockdown, and overexpression). Research has demonstrated that HSPA1L suppression significantly decreases sphere formation, while overexpression enhances it .

• Colony formation assays: Assessing clonogenic potential in relation to HSPA1L expression, which reveals the self-renewal capacity of cancer cells.

• Radiation resistance testing: Examining cell survival after γ-radiation exposure, as HSPA1L has been implicated in radiation resistance through the HSPA1L/IGF1Rβ/AKT pathway .

• Immunofluorescence co-localization studies: Using FITC-conjugated HSPA1L antibodies alongside other stem cell markers (CD44, CD133) to visualize spatial relationships.

When analyzing results, it's critical to consider that HSPA1L influences CSC properties through multiple pathways, including direct transcriptional regulation of β-catenin and enhancement of IGF1Rβ activation .

What are the key considerations when designing co-localization experiments using FITC-conjugated HSPA1L antibodies with other fluorescently labeled antibodies?

Designing effective co-localization experiments with FITC-conjugated HSPA1L antibodies requires careful planning to ensure reliable and interpretable results:

• Spectral compatibility: FITC emits green fluorescence (emission peak ~519nm), so partner fluorophores should have minimal spectral overlap. Recommended pairings include:

  • FITC (HSPA1L) + TRITC/Cy3 (red: IGF1Rβ)

  • FITC (HSPA1L) + Cy5/Alexa 647 (far-red: integrin αV)

• Sequential staining protocol: For optimal results when investigating the HSPA1L/IGF1Rβ/integrin αV triple complex:

  • Fix cells with 4% paraformaldehyde (15 minutes, room temperature)

  • Permeabilize with 0.1% Triton X-100 (10 minutes)

  • Block with 3% BSA (1 hour)

  • Apply FITC-conjugated HSPA1L antibody (1:50-1:500 dilution, based on optimization)

  • Apply compatible conjugated antibodies for other targets

  • Counterstain nuclei with DAPI

• Controls: Include single-stained samples for each antibody to confirm specificity and rule out cross-reactivity or bleed-through.

• Quantitative analysis: Use co-localization coefficients (Pearson's, Manders') to quantify spatial relationships between HSPA1L and interacting partners.

• Subcellular localization assessment: HSPA1L functions both in cytoplasm (for IGF1Rβ complex formation) and nucleus (for β-catenin transcriptional regulation) . Z-stack confocal imaging provides the most accurate representation of these distinct pools.

• Confirming functional associations: Complement imaging with biochemical approaches like proximity ligation assay (PLA) or co-immunoprecipitation to validate physical interactions suggested by co-localization .

How can HSPA1L antibody be effectively utilized to study its dual role in IGF1Rβ activation and β-catenin transcriptional regulation?

HSPA1L's dual functionality presents a unique research opportunity requiring specialized approaches to distinguish between its cytoplasmic role (IGF1Rβ activation) and nuclear role (β-catenin transcription):

• Subcellular fractionation coupled with immunoblotting:

  • Separate nuclear and cytoplasmic fractions

  • Probe each fraction with HSPA1L antibody

  • Compare relative abundance between compartments

  • Correlate with IGF1Rβ phosphorylation status and β-catenin levels

• Chromatin immunoprecipitation (ChIP) assay:

  • Use HSPA1L antibodies to immunoprecipitate protein-DNA complexes

  • Analyze binding to β-catenin promoter regions using specific primers

  • Recommended primers should target the region around exon 1 of β-catenin, which has been identified as a HSPA1L binding site

• Immunofluorescence with co-localization analysis:

  • FITC-conjugated HSPA1L antibody

  • Complementary antibodies for IGF1Rβ, integrin αV (cytoplasmic function)

  • Antibodies for transcription machinery components (nuclear function)

  • Confocal microscopy with Z-stack analysis

• Mutant constructs with compartment-specific targeting:

  • Nuclear export signal (NES) mutants to restrict HSPA1L to cytoplasm

  • Nuclear localization signal (NLS) mutants to enhance nuclear localization

  • Compare effects on IGF1Rβ phosphorylation versus β-catenin expression

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ

  • Distinguish between HSPA1L-IGF1Rβ-integrin αV complex and HSPA1L-DNA interactions

What are the optimal fixation and permeabilization protocols for HSPA1L immunofluorescence staining with FITC-conjugated antibodies?

The detection of HSPA1L using FITC-conjugated antibodies requires careful optimization of fixation and permeabilization conditions to preserve both antigenicity and fluorescence signal:

Recommended fixation protocols based on sample type:

Permeabilization optimization:

For detecting the dual localization of HSPA1L (cytoplasmic and nuclear), permeabilization conditions must be carefully balanced:

• For predominantly cytoplasmic HSPA1L detection (IGF1Rβ complex):

  • 0.1% Triton X-100 in PBS, 10 minutes at room temperature

  • Alternative: 0.5% Saponin in PBS, 15 minutes at room temperature

• For enhanced nuclear HSPA1L detection (β-catenin transcriptional role):

  • 0.5% Triton X-100 in PBS, 15-20 minutes at room temperature

  • Add a nuclear antigen retrieval step: 10mM sodium citrate buffer (pH 6.0) for 10-20 minutes at 95°C

• For dual detection experiments:

  • Sequential approach: Start with gentler permeabilization (0.1% Triton X-100, 10 minutes)

  • Optional nuclear antigen retrieval if nuclear signal is weak

  • Use higher antibody concentration (1:50-1:100 dilution) than typically used for single-location detection

Based on published protocols, the optimal antibody incubation for FITC-conjugated HSPA1L antibodies is overnight at 4°C in a humidity chamber protected from light to prevent photobleaching .

What are the recommended dilutions and incubation conditions for FITC-conjugated HSPA1L antibodies across different applications?

Optimal dilutions and conditions for FITC-conjugated HSPA1L antibodies vary by application. While specific data for FITC-conjugated versions is limited, these recommendations are extrapolated from unconjugated antibody protocols with adjustments for fluorescence preservation:

Critical optimization notes:

• Titration experiment: Always perform a titration experiment (1:20, 1:50, 1:100, 1:200, 1:500) with positive controls (e.g., HepG2 cells, which express high levels of HSPA1L) .

• Signal-to-noise ratio: FITC can produce background autofluorescence, especially in tissue samples. Include blocking steps with 10% normal serum from the same species as the secondary antibody (if using indirect methods).

• Photobleaching prevention: FITC is relatively prone to photobleaching. Use antifade mounting media containing anti-photobleaching agents like ProLong Gold or DABCO.

• Storage of stained samples: Store at 4°C in the dark; FITC signal typically remains stable for 1-2 weeks under proper storage conditions.

• Multiplexing considerations: When combining with other fluorophores, analyze samples promptly to minimize differential photobleaching rates between fluorophores .

How should researchers optimize HSPA1L detection in dual cytoplasmic/nuclear localization studies?

HSPA1L's dual localization presents unique challenges for comprehensive detection. Research has shown that HSPA1L functions both in the cytoplasm (in complex with IGF1Rβ and integrin αV) and in the nucleus (binding to the β-catenin promoter) . Optimizing detection of both pools requires specific technical approaches:

Recommended optimization protocol:

• Fixation optimization:

  • Test parallel samples with different fixatives:

    • 4% paraformaldehyde (preserves cytoplasmic structures)

    • Methanol/acetone (often enhances nuclear antigen access)

  • Compare results to determine if one fixative favors a particular localization

• Permeabilization gradient:

  • Prepare a series of cells/tissues with increasing permeabilization strength:

    • 0.1% Triton X-100 (5 minutes) - Mild

    • 0.3% Triton X-100 (10 minutes) - Moderate

    • 0.5% Triton X-100 (15 minutes) - Strong

  • Evaluate which condition best reveals both localizations

• Antigen retrieval for nuclear detection:

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

  • Enzymatic retrieval with proteinase K (very mild conditions)

  • Compare with and without retrieval to determine impact on nuclear signal

• Confocal microscopy with Z-stack acquisition:

  • Capture multiple focal planes (0.5-1μm steps)

  • Generate maximum intensity projections and orthogonal views

  • Use nuclear counterstain (DAPI) to clearly delineate nuclear boundaries

• Quantitative analysis:

  • Measure nuclear-to-cytoplasmic ratio of HSPA1L signal

  • Use cell fractionation followed by Western blot as validation

  • Compare results under different experimental conditions

Validation approaches:

• Use nuclear-targeted or cytoplasm-restricted HSPA1L mutants as controls

• Perform parallel chromatin immunoprecipitation to confirm nuclear binding

• Co-localize with known markers (IGF1Rβ for cytoplasmic, histones for nuclear) .

What are common issues encountered with FITC-conjugated HSPA1L antibodies and how can they be resolved?

Researchers working with FITC-conjugated HSPA1L antibodies may encounter several technical challenges. Here are common issues and their solutions:

1. High background/non-specific staining:

• Problem: Diffuse green fluorescence throughout samples without distinct HSPA1L pattern.

• Solutions:

  • Increase blocking stringency (5-10% normal serum, 1-2 hours)

  • Add 0.1-0.3% Triton X-100 to blocking buffer

  • Include additional blocking with 10mg/ml BSA

  • Try different blocking agents (normal serum, BSA, casein, or commercial blockers)

  • Increase washing steps (5 washes, 5 minutes each)

2. Weak or absent nuclear HSPA1L signal:

• Problem: Only cytoplasmic HSPA1L visible despite expecting nuclear localization.

• Solutions:

  • Add heat-induced epitope retrieval step (citrate buffer, pH 6.0)

  • Increase permeabilization (0.5% Triton X-100, 20 minutes)

  • Extend primary antibody incubation (48 hours at 4°C)

  • Reduce fixation time to prevent overfixation

  • Optimize antibody concentration (try higher concentration)

3. Photobleaching during analysis:

• Problem: FITC signal fades rapidly during microscopic examination.

• Solutions:

  • Use anti-fade mounting medium (ProLong Gold, VECTASHIELD)

  • Minimize exposure to excitation light

  • Capture images immediately after preparation

  • Consider spectral imaging to collect signal with minimal exposure

  • Use newer generation anti-fade reagents containing anti-oxidants

4. Poor co-localization with interacting partners:

• Problem: Expected co-localization of HSPA1L with IGF1Rβ or integrin αV not observed.

• Solutions:

  • Optimize fixation to preserve protein complexes (try mild fixatives)

  • Use proximity ligation assay as alternative approach

  • Try sequential staining protocol

  • Validate antibody specificity independently

  • Consider using split-fluorescent protein approaches for live cell studies

5. Inconsistent staining pattern between experiments:

• Problem: Variable HSPA1L staining patterns across experimental replicates.

• Solutions:

  • Standardize fixation time precisely

  • Prepare fresh fixatives for each experiment

  • Control for cell confluence and growth conditions

  • Include positive controls (HepG2 cells)

  • Document lot-to-lot antibody variations

How can researchers accurately interpret HSPA1L expression patterns in relation to cancer stem cell characteristics?

Accurate interpretation of HSPA1L expression patterns is crucial for understanding its role in cancer stem cells. Based on published research findings, here is a framework for interpretation:

1. Establishing baseline expression patterns:

• HSPA1L shows heterogeneous expression in cancer cell populations

• Higher expression correlates with ALDH1high cells (established CSC marker)

• Co-expression with stem cell transcription factors (Sox2, Oct4, Nanog)

• Often co-expressed with CSC surface markers (CD133, CD44)

2. Quantitative assessment approaches:

• Use digital image analysis software to quantify signal intensity

• Establish H-score or intensity thresholds based on control samples

• Compare nuclear-to-cytoplasmic ratio across different cell populations

• Correlate with other CSC markers using multiparameter flow cytometry

3. Functional correlation matrix:

4. Context-dependent interpretation:

• In epithelial cancers: co-localize with EMT markers (vimentin, N-cadherin)

• In therapy-resistant populations: correlate with radiation/drug response

• In invasion/metastasis: assess relationship to cell motility markers

• In cell cycle phases: determine if expression is cell-cycle dependent

5. Experimental validation framework:

• Manipulate HSPA1L levels (siRNA knockdown, overexpression)

• Measure resulting changes in CSC properties (ALDH activity, sphere formation)

• Assess therapeutic response before/after HSPA1L modification

• Correlate with patient outcomes in clinical samples

What controls should be included when validating FITC-conjugated HSPA1L antibody specificity?

Proper validation of FITC-conjugated HSPA1L antibody specificity is essential for generating reliable research data. A comprehensive validation strategy should include:

1. Essential negative controls:

• Secondary antibody-only control (if using indirect detection method)

• Isotype control antibody (matched to HSPA1L antibody host and isotype)

• HSPA1L-knockdown cells (siRNA or CRISPR/Cas9)

• Blocking peptide competition (pre-incubation with immunizing peptide)

• Non-expressing tissue/cell line (based on RNA-seq or proteomics data)

2. Positive controls:

• Known HSPA1L-expressing cells (HepG2, MCF-7, HeLa)

• Heat-shocked cells (HSPA1L is stress-inducible)

• HSPA1L-overexpressing cells (transfected with expression vector)

• Tissues with documented expression (testis, certain cancer tissues)

3. Specificity validation experiments:

• Western blot correlation: Confirm that IF pattern corresponds to WB band at expected MW (70 kDa)

• Peptide competition: Pre-incubate antibody with immunizing peptide before staining

• Cross-reactivity assessment: Test against related HSP70 family members

• Multiple antibody validation: Compare staining pattern with alternative HSPA1L antibody clones

• Orthogonal validation: Correlate protein expression with mRNA levels (RNA-FISH or RT-PCR)

4. Fluorophore-specific controls:

• Autofluorescence control: Unstained sample to assess background

• Spectral overlap control: When multiplexing with other fluorophores

• Photobleaching rate determination: Serial imaging under standard conditions

• pH sensitivity test: FITC fluorescence can be affected by environmental pH

5. Documentation requirements:

• Antibody catalog number and lot number

• Complete staining protocol with all parameters

• Microscope settings (exposure time, gain, etc.)

• Image processing steps

• Quantification method

An exemplary validation approach would combine siRNA knockdown of HSPA1L with Western blot and immunofluorescence to demonstrate parallel reduction in signal across methods, confirming specificity of the FITC-conjugated antibody .