HSPA1L Antibody, Biotin conjugated

What is HSPA1L and what cellular functions does it serve?

HSPA1L (also known as HSP-A1L, HSPA1-L, HSP70-HOM, or Hum70t) is a member of the heat shock protein 70 family. It functions in several cellular processes including signal transduction, infection immunity, developmental processes, and pulmonary functions . The protein has a calculated and observed molecular weight of 70 kDa . As a heat shock protein, it plays critical roles in protein folding, cellular stress responses, and proteostasis maintenance, though its specific functions may differ from other HSP70 family members.

How does biotin conjugation enhance HSPA1L antibody utility in research applications?

Biotin conjugation significantly enhances HSPA1L antibody utility by enabling signal amplification through the high-affinity interaction between biotin and streptavidin/avidin. This conjugation allows for increased sensitivity in detection systems, particularly in ELISA applications . The biotin-streptavidin interaction provides a versatile platform for detection through various secondary reagents, making biotin-conjugated antibodies particularly valuable in multi-step detection protocols and when working with samples containing low target protein concentrations.

What species reactivity can researchers expect with HSPA1L antibodies?

According to the available data, HSPA1L antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibodies have also been cited for reactivity with bovine samples . This cross-species reactivity allows for comparative studies across different model organisms, though researchers should verify specific reactivity patterns for their particular experimental system as performance can vary between antibody preparations and applications.

What are the recommended dilutions for biotin-conjugated HSPA1L antibodies across applications?

The optimal dilution ranges for biotin-conjugated HSPA1L antibodies vary by application:

It is essential to titrate the antibody in each specific testing system to determine optimal conditions, as sample type and experimental conditions can significantly impact performance .

What antigen retrieval methods are most effective for HSPA1L detection in fixed tissues?

For optimal HSPA1L detection in paraffin-embedded tissue sections, heat-mediated antigen retrieval using EDTA buffer (pH 8.0) has proven effective . Alternative approaches include using TE buffer at pH 9.0 or citrate buffer at pH 6.0 . The choice of antigen retrieval method can significantly impact detection sensitivity and should be optimized based on tissue type and fixation conditions. Enzyme antigen retrieval using commercial reagents like IHC enzyme antigen retrieval reagent (AR0022) has also been successfully employed in some protocols .

What positive controls are recommended for validating HSPA1L antibody experiments?

Based on published validation data, the following samples serve as effective positive controls:

Using these validated positive controls enables researchers to confirm antibody performance before proceeding with experimental samples.

How can biotin-conjugated HSPA1L antibodies be utilized in co-localization studies?

Biotin-conjugated HSPA1L antibodies are particularly valuable for co-localization studies due to their compatibility with various detection systems. For immunofluorescence applications, researchers should first optimize the antibody dilution (typically starting at 1:50-1:500) and then use streptavidin conjugated to a fluorophore distinct from other detection channels. When designing multi-color experiments, consider using far-red streptavidin conjugates to avoid spectral overlap with common fluorophores like FITC or TRITC. Successful immunofluorescence detection has been demonstrated in cell lines such as HepG2 , making these viable models for co-localization studies involving HSPA1L.

What techniques can be employed to quantify HSPA1L expression levels using biotin-conjugated antibodies?

Quantification of HSPA1L expression can be accomplished through several techniques using biotin-conjugated antibodies:

• Quantitative Western blotting: Using standard curves with recombinant HSPA1L protein at known concentrations alongside experimental samples at dilutions of 1:500-1:2000 .

• ELISA-based quantification: Biotin-conjugated HSPA1L antibodies are particularly suited for quantitative ELISA protocols, where streptavidin-HRP provides sensitive and specific detection .

• Quantitative immunohistochemistry: Digital image analysis of IHC staining intensity using antibody dilutions of 1:100-1:400 , with appropriate normalization to housekeeping proteins.

• Flow cytometry: For cellular quantification of HSPA1L levels, with careful selection of appropriate isotype controls .

How do HSPA1L expression patterns differ between normal and pathological tissues?

Based on immunohistochemical analyses, HSPA1L demonstrates tissue-specific expression patterns. In pathological contexts, significant HSPA1L expression has been detected in human ovarian cancer tissue, human endometrial cancer tissue, and human breast cancer tissue . In normal tissues, HSPA1L is notably expressed in mouse and rat testis tissues and liver tissues . These differential expression patterns highlight the potential significance of HSPA1L in both normal physiology and pathological processes, particularly in reproductive tissues and cancers of reproductive organs.

What are common causes of background staining when using biotin-conjugated HSPA1L antibodies?

Several factors can contribute to background staining when using biotin-conjugated HSPA1L antibodies:

• Endogenous biotin: Tissues like liver, kidney, and brain contain high levels of endogenous biotin that can interfere with detection. Pre-blocking with avidin/biotin blocking kits is recommended.

• Insufficient blocking: Increase blocking time or concentration of blocking agent (10% goat serum has been successfully used in validated protocols) .

• Antibody concentration: Excessive antibody concentration can increase background. Titrate carefully, starting with recommended dilutions of 1:100-1:400 for IHC applications .

• Cross-reactivity: Due to the conserved nature of heat shock proteins, ensure specific detection of HSPA1L versus related family members by validating with appropriate controls.

• Detection system sensitivity: Adjust the development time when using DAB as a chromogen to optimize signal-to-noise ratio .

How should biotin-conjugated HSPA1L antibodies be stored for optimal performance?

For maximum stability and performance, biotin-conjugated HSPA1L antibodies should be stored according to manufacturer recommendations. Typically, this involves:

• Long-term storage: Store at -20°C for up to one year from the date of receipt .

• After reconstitution: Store at 4°C for up to one month, or aliquot and store frozen at -20°C for up to six months .

• Avoid repeated freeze-thaw cycles: This can significantly degrade antibody performance and lead to inconsistent results .

• Some preparations contain stabilizers: For example, storage buffers may include PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 , which helps maintain antibody integrity.

How do biotin-conjugated HSPA1L antibodies compare to other conjugated forms?

Various conjugated forms of HSPA1L antibodies are available for different applications:

Biotin conjugation offers particular advantages in sensitivity and versatility compared to direct enzyme or fluorophore conjugates, though it requires additional detection steps.

What protocols have been validated for detecting HSPA1L in different subcellular compartments?

Validated protocols for detecting HSPA1L in different subcellular compartments include:

• For cytoplasmic and nuclear detection: Immunocytochemistry using fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 is effective. Antibody dilutions of 1:50-1:500 are recommended for immunofluorescence applications .

• For membrane-associated HSPA1L: Membrane fractionation followed by Western blotting using 1:500-1:2000 antibody dilution has been validated .

• For tissue-specific localization: Immunohistochemistry using paraffin-embedded sections with EDTA buffer (pH 8.0) antigen retrieval, followed by incubation with 2 μg/ml antibody overnight at 4°C provides specific subcellular localization information .

How can researchers differentiate between HSPA1L and other closely related heat shock proteins?

Differentiating between HSPA1L and related heat shock proteins requires careful experimental design:

• Antibody selection: Use antibodies raised against unique epitopes of HSPA1L. The immunogen information should be consulted to ensure specificity .

• Validation with knockdown/knockout controls: One publication has demonstrated successful use of HSPA1L antibodies in knockdown/knockout systems , which provides the strongest evidence for specificity.

• Western blot analysis: Although HSPA1L shares the 70 kDa molecular weight with other HSP70 family members, subtle migration differences in high-resolution gels can help distinguish between related proteins .

• Expression pattern analysis: Compare detection patterns with known tissue-specific expression profiles of different HSP70 family members, as these proteins often show differential tissue expression despite sequence similarity.