HSPA1L Antibody, HRP conjugated

What is HSPA1L and what is its biological function?

HSPA1L is a 70kDa heat shock protein that belongs to the heat shock protein 70 family. It functions in conjunction with other heat shock proteins to stabilize existing proteins against aggregation and mediate the folding of newly translated proteins in the cytosol and organelles. The gene is located in the major histocompatibility complex class III region, in a cluster with two closely related genes which also encode isoforms of the 70kDa heat shock protein . HSPA1L plays important roles in various physiological processes, including protection of proximal tubular cells in diabetic kidney disease and maintenance of sperm motility and quality .

What are the recommended dilutions for HSPA1L antibody and HRP-conjugated secondary antibody in Western blot experiments?

Always perform a dilution series optimization for your specific experimental conditions and antibody.

For which applications can HSPA1L antibody be used?

HSPA1L antibodies have been validated for multiple applications:

The same antibody may show different performance in different applications, so validation for each specific application is recommended.

What is the molecular weight of HSPA1L protein and how should I interpret Western blot results?

HSPA1L has both a calculated and observed molecular weight of 70 kDa . When performing Western blot analysis, you should expect to see a distinct band at approximately 70 kDa. If you observe multiple bands, additional validation may be necessary to confirm specificity. The high sequence homology among HSP70 family members can sometimes lead to cross-reactivity, so antibody selection and validation are crucial for accurate HSPA1L detection .

How does HSPA1L expression differ between various human tissues, and what are the implications for experimental design?

HSPA1L demonstrates tissue- and cell-type-specific expression patterns that should be considered when designing experiments:

• Testis/Sperm: HSPA1L is expressed on sperm post-acrosome and midpiece regions . Testis can serve as a positive control for HSPA1L detection .

• Kidney: HSPA1L is expressed in proximal tubular cells and plays a protective role against metabolic stress .

• Normal tissues: HSPA1L shows distinct expression patterns that differ from other HSP70 family members, suggesting tissue-specific roles .

When designing experiments:

• Include appropriate positive controls based on known expression patterns

• Consider cell-type heterogeneity within tissue samples

• Use sufficient sample size to account for individual variation

• Compare expression with other HSP70 family members to establish specificity

What is the difference between HSPA1L and other HSP70 family members in terms of function and expression patterns?

The HSP70 family includes several members with distinct properties and functions:

HSPA1L forms specific complexes with vaspin and GRP78 in proximal tubular cells, protecting against metabolic stress-induced injuries . In sperm, HSPA1L antibody inhibits motility in blocking experiments, while HSPA9 antibody shows no significant effect, highlighting functional differences between family members .

How do experimental conditions affect HSPA1L detection?

Several experimental conditions can influence HSPA1L detection:

• Stress induction: ER stressors like tunicamycin and thapsigargin or metabolic stressors like palmitate can alter HSPA1L complex formation and localization .

• Sample preparation: HSPA1L antibody is typically lyophilized in PBS buffer with 2% sucrose. Add 50 μL of distilled water for a final concentration of 1 mg/mL .

• Storage conditions: Aliquot and store at -20°C or below. Avoid multiple freeze-thaw cycles .

• Antigen retrieval for IHC: For paraffin-embedded tissues, suggested antigen retrieval with TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 may be used .

• Protein-protein interactions: HSPA1L forms complexes with other proteins like vaspin and GRP78, which may affect epitope accessibility .

A systematic approach to optimizing these conditions will ensure consistent and reliable HSPA1L detection.

What approaches can be used to study HSPA1L interactions with other proteins?

HSPA1L forms functional complexes with several proteins. These interactions can be studied using multiple approaches:

• Co-immunoprecipitation: The complex formation of vaspin with HSPA1L and GRP78 was observed in proximal tubular cells after overexpression of tagged proteins .

• Expression systems: HEK293T cells can be used for protein interaction studies due to better scale-up efficiency for protein preparation in immunoprecipitation experiments .

• Clathrin-mediated endocytosis studies: HSPA1L and GRP78 both bind to clathrin heavy chain and are involved in endocytosis, suggesting functional interactions that can be studied through endocytosis assays .

• Proximity Ligation Assay: This technique can detect protein-protein interactions in situ and has been validated for HSPA1L antibodies .

• Functional studies: Overexpression of HSPA1L dissolved organelle stresses, especially autophagy impairment, providing functional evidence of its interactions with stress response pathways .

What are common causes of non-specific binding when using HSPA1L antibody, and how can they be minimized?

Non-specific binding can significantly impact experimental results when using HSPA1L antibodies:

Common causes:

• Cross-reactivity with other HSP70 family members (91% homology between HSPA1L and HSPA1A)

• Inappropriate blocking conditions

• Excessive antibody concentration

• Inadequate washing steps

Solutions:

• Antibody validation: Confirm specificity using Western blot against multiple HSP70 family members .

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity .

• Optimization: Use recommended dilutions (1 μg/mL for Western blot) .

• Blocking optimization: Test different blocking agents (BSA, normal serum from the same species as the secondary antibody).

• Washing stringency: Include sufficient washing steps with appropriate detergent concentration.

• Controls: Include negative controls lacking primary antibody and positive controls with known HSPA1L expression.

How can I validate the specificity of my HSPA1L antibody?

Rigorous validation ensures reliable experimental results. Several approaches are recommended:

• Western blot against recombinant proteins: Test cross-reactivity with related HSP70 family members. For example, test against "human recombinant HSPA2-GST fusion protein, HSPA6, HSPA1 and HSPA8" .

• Peptide competition assay: Pre-incubate the antibody "with mass excess of the peptide antigen (1 μg) used for immunization" and confirm signal reduction .

• Tissue controls: Use tissues with known HSPA1L expression patterns. Testes serve as positive control for HSPA2, while prostate can be used for HSPA1 proteins .

• Multiple antibody comparison: Compare results from antibodies recognizing different epitopes of HSPA1L.

• Immunoprecipitation-mass spectrometry: Confirm pulled-down protein identity through mass spectrometry.

• Application-specific validation: An antibody may perform well in one application but not in others, so validation should be performed for each intended application.

What are potential causes of weak or no signal when using HSPA1L antibody in Western blot?

Troubleshooting weak or absent signals requires a systematic approach:

Technical issues:

• Antibody concentration: Ensure proper dilution (1 μg/mL recommended) or 1:500-1:2000 .

• Secondary antibody dilution: HRP conjugated secondary antibody should be diluted 1:50,000-100,000 .

• Transfer efficiency: Optimize transfer conditions for the 70 kDa molecular weight of HSPA1L.

• Detection sensitivity: Use enhanced chemiluminescence systems appropriate for your expected signal strength.

Biological issues:

• Expression level: HSPA1L shows cell-type-specific expression patterns . Confirm HSPA1L expression in your sample type.

• Sample preparation: Ensure proper sample handling to prevent protein degradation. Antibody is typically purified by peptide affinity chromatography .

• Protein extraction: Use lysis buffers that effectively extract HSPA1L while preserving epitope integrity.

• Storage conditions: Store antibody as recommended (aliquot and store at -20°C, avoid freeze-thaw cycles) .

How does sample preparation affect the detection of HSPA1L?

Proper sample preparation is critical for successful HSPA1L detection:

• Antibody reconstitution: For lyophilized antibody, add 50 μL of distilled water for a final concentration of 1 mg/mL .

• Lysate preparation: Include protease inhibitors to prevent degradation. For Western blot samples, typical loading amounts range from 10-30 μg total protein.

• Application-specific considerations:

  • Western blot: Denaturing conditions (SDS-PAGE) are typically used

  • Immunoprecipitation: 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Immunohistochemistry: Formalin-fixed paraffin-embedded tissues require proper antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

• Tissue-specific protocols: For sperm samples, specialized extraction protocols may be necessary to study HSPA1L in post-acrosome and midpiece regions .

• Subcellular fractionation: When studying HSPA1L's role in endocytosis or its association with clathrin , appropriate fractionation protocols should be employed.

How can HSPA1L antibody be used to study stress responses in cellular models?

HSPA1L plays important roles in cellular stress responses, particularly in protection against organelle stress:

Experimental approaches:

• Stress induction models: Use ER stressors (tunicamycin, thapsigargin) or metabolic stressors (palmitate) to induce cellular stress in models such as proximal tubular cells (HK2 cells) .

• Expression analysis: Monitor HSPA1L expression changes using Western blot (1 μg/mL antibody concentration) during stress responses.

• Localization studies: Use immunofluorescence (IF/ICC dilution 1:50-1:500) to examine changes in subcellular localization during stress responses.

• Co-localization with stress markers: Examine HSPA1L alongside ER stress markers (GRP78, phosphorylated eIF2α, CHOP) and autophagy markers (p62) .

• Functional analysis: Study the protective effects of HSPA1L through overexpression or knockdown experiments. HSPA1L overexpression has been shown to dissolve organelle stresses, especially autophagy impairment .

• Protein complex formation: Examine how stress affects HSPA1L complex formation with partners like vaspin and GRP78 using co-immunoprecipitation .

What are the considerations for using HSPA1L antibody in immunoprecipitation experiments?

Immunoprecipitation is valuable for studying HSPA1L's interactions with other proteins:

Protocol considerations:

• Antibody amount: Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Cell line selection: HEK293T cells provide better scale-up efficiency for protein preparation in immunoprecipitation studies .

• Expression systems: Consider using tagged overexpression systems (e.g., FLAG-tagged constructs) to enhance detection and specificity .

• Complex formation analysis: HSPA1L forms complexes with vaspin and GRP78 that can be detected by co-immunoprecipitation .

• Controls: Include isotype-matched irrelevant antibodies or no-antibody controls to assess non-specific binding.

• Washing conditions: Optimize washing stringency to maintain specific interactions while reducing background.

• Detection methods: Use sensitive detection methods appropriate for the expected abundance of your target and interacting proteins.

How should HSPA1L antibody be used in immunohistochemistry of paraffin-embedded tissues?

Immunohistochemistry (IHC) allows visualization of HSPA1L expression in tissue contexts:

Protocol recommendations:

• Antibody dilution: Use 1:100-1:400 or 1:10-1:500 dilution for IHC-P applications.

• Antigen retrieval: Perform antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

• Positive controls: Include appropriate positive controls such as testis tissue. For monoclonal antibody to HSPA1L, a concentration of 3 μg/ml has been validated on formalin-fixed paraffin-embedded human testis .

• Scoring system: Consider using a semi-quantitative scoring system based on staining intensity:

  • Strong staining: Bright/dark intensity visible at low magnification (40×)

  • Moderate staining: Definitely lighter than bright/dark, visible at low magnification (40×)

  • Weak staining: Visible at 100× magnification

  • No staining: Barely visible at high magnification (400×) or absent

• Independent validation: Have multiple investigators score samples independently to ensure reproducibility .

• Tissue-specific patterns: Note that HSPA1L shows cell-type-specific expression patterns that differ from other HSP70 family members .

What controls should be included when using HSPA1L antibody in immunofluorescence studies?

Proper controls ensure reliable interpretation of immunofluorescence results:

• Positive controls: Include cells or tissues known to express HSPA1L. HepG2 cells have been validated for HSPA1L immunofluorescence . In sperm, HSPA1L localizes to post-acrosome and midpiece regions .

• Negative controls: Include primary antibody omission controls to assess non-specific binding of secondary antibodies.

• Peptide competition controls: Pre-incubate antibody with immunizing peptide to confirm specificity .

• Dilution optimization: Test the recommended dilution range (1:50-1:500 for IF/ICC) to determine optimal signal-to-noise ratio.

• Co-localization controls: Include markers for subcellular compartments to verify localization patterns. For example, in studies of endocytosis, co-staining with clathrin may be informative .

• Fixation controls: Different fixation methods may affect epitope accessibility; optimize fixation conditions for your specific application.

• Isotype controls: Include an irrelevant antibody of the same isotype and concentration as the HSPA1L antibody.

How should I interpret changes in HSPA1L expression levels in disease models?

Changes in HSPA1L expression can provide insights into disease mechanisms:

• Kidney disease models: Reduced HSPA1L expression in proximal tubular cells may indicate increased vulnerability to:

  • ER stress

  • Autophagy impairment

  • Lysosomal membrane permeabilization

  • Cell death

• Reproductive biology: Decreased HSPA1L expression in spermatozoa correlates with reduced motility and poor sperm quality . HSPA1L antibody can inhibit sperm motility in functional blocking experiments .

• Age-related changes: Compared to young adult testes with normal spermatogenesis, HSPA1L shows decreased expression in elderly testis characterized by poor spermatogenesis .

• Tissue specificity: HSPA1L expression patterns differ significantly from other HSP70 family members, suggesting distinct functional roles . Changes should be interpreted in a tissue-specific context.

• Protein interactions: Consider how HSPA1L expression changes affect interactions with partners like vaspin and GRP78, which form functional complexes in certain cell types .

What is the significance of subcellular localization changes of HSPA1L?

HSPA1L's function is closely tied to its subcellular localization:

• Normal localization patterns:

  • In sperm: HSPA1L localizes to post-acrosome and midpiece regions

  • In proximal tubular cells: HSPA1L forms complexes with vaspin and GRP78

• Stress-induced changes: During metabolic stress or ER stress, HSPA1L localization and complex formation may change. Albumin overload enhances extracellular release of HSPA1L from proximal tubular cells .

• Endocytosis role: HSPA1L and vaspin complexes are internalized by clathrin-mediated endocytosis, indicating a role in vesicular trafficking .

• Functional implications: HSPA1L localization changes correlate with its ability to protect against organelle stress, particularly autophagy impairment and lysosomal membrane permeabilization .

• Comparison with other HSP70 members: Different HSP70 family members show distinct subcellular localization patterns, reflecting their specialized functions .

How does HSPA1L interact with other proteins, and how can these interactions be studied?

HSPA1L forms functional protein complexes that can be studied through various approaches:

Key interactions:

• Vaspin complex: During internalization into proximal tubular cells, vaspin forms a complex with HSPA1L and GRP78 .

• Clathrin association: Both HSPA1L and GRP78 bind to clathrin heavy chain and participate in endocytosis .

• Stress response machinery: HSPA1L interactions protect against ER stress, autophagy impairment, and lysosomal membrane permeabilization .

Study methods:

• Co-immunoprecipitation: Detects protein complexes in cell lysates. HEK293T cells provide better efficiency for protein preparation in immunoprecipitation studies .

• Overexpression systems: Plasmids expressing tagged proteins (e.g., 3xFLAG-Vaspin) can be used to enhance detection of interactions .

• Functional studies: Albumin overload enhances extracellular release of HSPA1L, providing a model to study how interactions change under stress conditions .

• Proximity Ligation Assay: Detects protein-protein interactions in situ with high sensitivity and has been validated for HSPA1L antibodies .

• Subcellular fractionation: Helps determine where within the cell specific interactions occur.

What is the relationship between HSPA1L expression and cellular stress responses?

HSPA1L plays critical roles in cellular stress responses, particularly in protecting organelle function:

• Protection against ER stress: HSPA1L-vaspin pathways ameliorate enhanced ER stress in proximal tubular cells. ER stressors (tunicamycin, thapsigargin) increase expressions of stress markers like GRP78, phosphorylated eIF2α, and CHOP .

• Autophagy regulation: Overexpression of HSPA1L dissolves organelle stresses, especially autophagy impairment. HSPA1L pathways prevent accumulation of p62, an indicator of impaired autophagy .

• Lysosomal membrane protection: HSPA1L-vaspin complex ameliorates obesity-induced lysosomal membrane permeabilization, inflammasome activation, and autophagy failure. This protects against cathepsin B leakage into cytosol .

• Stress-induced release: Albumin overload enhances extracellular release of HSPA1L, reducing intracellular levels and potentially compromising protective functions .

• Distinction from other HSP70 members: Unlike stress-inducible HSPA1, which is expressed at low levels in normal conditions and highly induced during stress, HSPA1L shows constitutive expression patterns in specific cell types .

This relationship suggests HSPA1L could be a potential therapeutic target for diseases involving organelle stress, particularly in kidney cells and reproductive tissues.