hsd11b1l Antibody

What is HSD11B1L and how does it differ from HSD11B1?

HSD11B1L (Hydroxysteroid 11-beta dehydrogenase 1-like) is a species-restricted member of the short-chain dehydrogenase/reductase (SDR) superfamily located on human chromosome 19p13.3. The gene spans approximately 7.5 kb and consists of 9 exons . It shows approximately 40% amino acid identity with human 11βHSD1, with strong conservation of key catalytic enzyme domains and cofactor binding sites .

Unlike HSD11B1, which functions as a NADP(H)-dependent reductase converting inactive cortisone to active cortisol and is widely expressed in liver, adipose tissue, adrenal, ovary, and decidua , HSD11B1L has been reported to have only weak substrate dehydrogenase activity for cortisol in vitro . Additionally, an important distinguishing feature is that HSD11B1L is absent in rodent genomes, suggesting it serves a unique function in humans and other species .

What is the tissue distribution pattern of HSD11B1L?

HSD11B1L demonstrates a distinctive expression pattern that differs from related dehydrogenases. Studies have detected moderate expression in total RNA from whole brain, with stronger expression in ovary, testis, and lung tissues . Weaker expression has been observed in human kidney . Comparative analysis with 11βHSD1 and 11βHSD2 has shown that HSD11B1L and 11βHSD1 have similar mRNA expression levels in the brain, while both are almost undetectable in human colon tissues where 11βHSD2 is highly expressed . This tissue-specific distribution pattern suggests specialized roles for HSD11B1L in reproductive and neurological functions.

What are the critical considerations when selecting an HSD11B1L antibody for research?

When selecting an HSD11B1L antibody, researchers should consider several key factors:

• Epitope specificity: Due to the presence of alternative splicing in exon 9 that generates two isoforms (A and B) with variable C-terminal ends , antibodies targeting different regions may produce varying results. C-terminal targeting antibodies like those described in search result are designed to recognize specific regions (amino acids 256-283 in the referenced antibody) .

• Species reactivity: Since HSD11B1L is absent in rodent genomes , antibodies should be validated specifically for human applications and any other target species of interest.

• Application compatibility: Verify that the antibody has been validated for your specific application (Western blot, immunohistochemistry, immunofluorescence) .

• Cross-reactivity: Consider potential cross-reactivity with the related HSD11B1 protein due to their 40% amino acid identity , and select antibodies that have been specifically tested for specificity.

• Clonality: Determine whether polyclonal antibodies (which recognize multiple epitopes) or monoclonal antibodies (which recognize a single epitope) are more appropriate for your specific research question .

How can I validate the specificity of an HSD11B1L antibody?

Robust validation of HSD11B1L antibodies can be achieved through several complementary approaches:

• Positive control samples: Use tissue or cell types known to express HSD11B1L, such as brain, ovary, testis, or lung tissue samples . Cell lines like U-251MG or K562 have been successfully used in previous studies .

• Western blot analysis: Confirm the specificity by checking if the antibody detects a protein of the expected molecular weight. The observed molecular weight may differ from the calculated molecular weight due to post-translational modifications.

• Blocking peptide experiments: Use the immunizing peptide (if available) to competitively block antibody binding, which should eliminate specific signals.

• Knock-down or knock-out controls: Where possible, use samples from knock-down (siRNA) or knock-out (CRISPR) experiments to confirm antibody specificity.

• Multiple antibody comparison: Use antibodies targeting different epitopes of HSD11B1L and compare results to confirm specificity.

• Multiple detection methods: Compare results from different techniques such as Western blotting, immunohistochemistry, and immunofluorescence to ensure consistent detection patterns .

What are the optimal protocols for Western blot analysis using HSD11B1L antibodies?

For optimal Western blot analysis with HSD11B1L antibodies, the following protocol is recommended:

• Sample preparation:

  • For cell lines (e.g., K562), use approximately 35 μg of total protein per lane

  • Include positive controls from tissues known to express HSD11B1L (brain, ovary, testis, lung)

• SDS-PAGE separation:

  • Use gradient gels (4-12% or 4-20%) to improve separation

  • Include molecular weight markers that span the expected size range

• Transfer conditions:

  • Transfer to PVDF membrane using standard wet transfer protocols

  • Transfer overnight at low voltage (30V) for larger proteins

• Blocking:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute HSD11B1L antibody according to manufacturer recommendations (typically 1:200-1:1000)

  • Incubate overnight at 4°C with gentle rocking

• Secondary antibody:

  • Use appropriate HRP-conjugated anti-rabbit secondary antibody

  • Typically diluted 1:5000-1:10000

  • Incubate for 1-2 hours at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) for detection

  • Expose to X-ray film or use digital imaging system

• Expected results:

  • HSD11B1L should be detected at the appropriate molecular weight, though the observed weight may differ from the calculated weight due to post-translational modifications

This protocol has been successfully used to detect HSD11B1L in K562 cell line lysates .

How should I optimize immunohistochemistry protocols for HSD11B1L detection?

Optimizing immunohistochemistry (IHC) protocols for HSD11B1L detection requires careful attention to several key steps:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • Human prostate carcinoma has been successfully used for HSD11B1L IHC validation

• Antigen retrieval:

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Heat in a pressure cooker or microwave until boiling, then maintain at sub-boiling temperature for 10-20 minutes

• Blocking:

  • Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 15 minutes

  • Block non-specific binding with 5% normal goat serum in PBS for 1 hour

• Primary antibody:

  • Dilute HSD11B1L antibody according to manufacturer recommendations

  • Incubate overnight at 4°C in a humidified chamber

• Secondary antibody and detection:

  • Use peroxidase-conjugated secondary antibody

  • Develop with DAB (3,3'-diaminobenzidine) substrate

  • Counterstain with hematoxylin

• Controls:

  • Include negative controls (omitting primary antibody)

  • Include positive controls (tissues known to express HSD11B1L such as brain, ovary, testis, or lung)

• Evaluation:

  • Assess staining pattern and intensity

  • Note that clinical relevance of HSD11B1L IHC staining patterns requires further evaluation

This approach has been documented to successfully detect HSD11B1L in human prostate carcinoma tissue sections .

What are the best practices for immunofluorescence detection of HSD11B1L?

For optimal immunofluorescence (IF) detection of HSD11B1L, follow these best practices:

• Cell preparation:

  • U-251MG cells have been successfully used for IF detection of HSD11B1L

  • Grow cells on glass coverslips or chamber slides to 70-80% confluence

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with 5% normal goat serum in PBS for 1 hour at room temperature

• Primary antibody:

  • Dilute HSD11B1L antibody according to manufacturer recommendations (typically 1:200-1:800 for IF applications)

  • Incubate overnight at 4°C in a humidified chamber

• Secondary antibody:

  • Use fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488-conjugated goat anti-rabbit IgG)

  • Typically diluted 1:500-1:1000

  • Incubate for 1-2 hours at room temperature in the dark

• Nuclear counterstaining:

  • Counterstain nuclei with DAPI (4′,6-diamidino-2-phenylindole)

  • Incubate for 5-10 minutes at room temperature

• Mounting and imaging:

  • Mount using antifade mounting medium

  • Image using confocal microscopy for optimal resolution

• Controls:

  • Include negative controls (omitting primary antibody)

  • Include positive controls (cell types known to express HSD11B1L)

This protocol has been successfully implemented for HSD11B1L detection in U-251MG cells .

How do HSD11B1L isoforms affect antibody selection and experimental design?

HSD11B1L has two known isoforms resulting from alternative RNA splicing in exon 9, generating A and B forms with variable C-terminal ends . This structural complexity introduces several important considerations for antibody selection and experimental design:

• Antibody epitope location:

  • Antibodies targeting the C-terminal region (like amino acids 256-283) may differentially recognize the A and B isoforms

  • For comprehensive detection of all isoforms, consider antibodies targeting conserved regions

• mRNA analysis design:

  • PCR primer design should account for the two alternate ATG start sites in exons 2 and 3

  • Consider using isoform-specific primers to differentiate between variants

• Expression patterns:

  • The relative abundance of each isoform may vary across different tissues

  • Brain tissue may express different isoform ratios compared to reproductive tissues

  • Design experiments to evaluate isoform-specific expression patterns

• Functional studies:

  • Different isoforms may have distinct subcellular localizations or functional properties

  • Consider isoform-specific knockdown or overexpression studies to delineate functions

• Protein size variation:

  • Western blot analysis should account for potential size differences between isoforms

  • Optimize gel conditions to resolve closely migrating isoform bands

Understanding these isoform considerations is crucial for accurate interpretation of experimental results and avoiding false negative findings that might occur if an antibody fails to detect a specific isoform.

What are the challenges in cross-species applications of HSD11B1L antibodies?

The absence of HSD11B1L in rodent genomes presents significant challenges for cross-species applications of HSD11B1L antibodies:

• Species-restricted expression pattern:

  • HSD11B1L is absent in all rodent genomes , limiting the use of common laboratory animal models (mice, rats)

  • Researchers should be aware that studies in these models will not represent HSD11B1L biology

• Alternative model selection:

  • For in vivo studies, researchers must select appropriate non-rodent models where HSD11B1L is expressed

  • Sheep have been used as an alternative model system for studying HSD11B1L expression patterns

• Antibody specificity across species:

  • Antibodies developed against human HSD11B1L may have variable reactivity with orthologs from other species

  • Validation in each target species is essential before experimental use

• Evolutionary implications:

  • The species-restricted nature of HSD11B1L suggests it may serve specialized functions in higher mammals

  • Comparative studies between species with and without HSD11B1L may provide insights into its evolutionary significance

• Functional compensation:

  • In species lacking HSD11B1L, other enzymes may compensate for its function

  • Understanding these compensatory mechanisms is important for translational research

These challenges highlight the importance of careful model selection and antibody validation when designing experiments to study HSD11B1L biology across different species.

What are common problems encountered when using HSD11B1L antibodies and how can they be addressed?

Researchers often encounter several challenges when working with HSD11B1L antibodies. Here are common problems and their solutions:

• Weak or no signal in Western blots:

  • Increase antibody concentration or extend incubation time

  • Optimize protein loading (35 μg/lane has been successful with K562 cell lysates)

  • Enhance sensitivity using amplified detection systems

  • Verify expression in your sample type by checking against tissue types known to express HSD11B1L (brain, ovary, testis, lung)

• High background in immunohistochemistry:

  • Increase blocking time or blocking agent concentration

  • Optimize antibody dilution (typically start with manufacturer's recommended range)

  • Reduce DAB development time

  • Include additional washing steps after primary and secondary antibody incubations

• Non-specific bands in Western blot:

  • Increase stringency of washing steps

  • Optimize blocking conditions

  • Use gradient gels for better protein separation

  • Perform peptide competition assays to identify specific bands

• Poor reproducibility:

  • Standardize sample collection and processing

  • Maintain consistent antibody storage conditions (-20°C long-term)

  • Avoid repeated freeze-thaw cycles of antibody

  • Use the same lot number of antibody for comparative experiments

• Difficulty detecting specific isoforms:

  • Select antibodies with epitopes specific to regions that differ between isoforms

  • Use higher resolution gel systems to separate closely migrating bands

  • Consider using isoform-specific RNA interference to confirm band identity

• Cross-reactivity with HSD11B1:

  • Validate antibody specificity using samples with known differential expression of HSD11B1 and HSD11B1L

  • Consider using tissues like colon, where HSD11B1L and HSD11B1 are almost undetectable while HSD11B2 is highly expressed

How should HSD11B1L antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of HSD11B1L antibodies is crucial for maintaining their performance and extending their useful life:

• Storage temperature:

  • Store at -20°C for long-term storage

  • For short-term storage (up to 1 week), refrigeration at 2-8°C is acceptable

• Aliquoting:

  • Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles

  • Aliquots of 10-20 μL are typically sufficient for individual experiments

• Storage buffer considerations:

  • Antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • This formulation helps maintain stability during freeze-thaw cycles

• Thawing protocol:

  • Thaw antibodies on ice or at 4°C

  • Avoid rapid warming to room temperature

  • Gently mix by flicking or inverting the tube (avoid vortexing)

• Shelf life:

  • Properly stored antibodies typically have a shelf life of 12 months from the date of shipment

  • Monitor performance over time with appropriate positive controls

• Shipping conditions:

  • Antibodies are typically shipped with blue ice

  • Upon receipt, immediately transfer to appropriate long-term storage conditions

• Contamination prevention:

  • Use sterile technique when handling antibodies

  • Avoid introducing bacteria or fungi that could degrade the antibody

Following these storage and handling guidelines will help ensure consistent performance of HSD11B1L antibodies across experiments and maximize their useful lifespan.