OR11L1 Antibody

What is OR11L1 and why do researchers study it?

OR11L1, also known as Olfactory Receptor Family 11 Subfamily L Member 1, belongs to the large family of olfactory receptors that function as G-protein coupled receptors involved in the detection of odor molecules and initiation of neuronal responses that trigger smell perception. Researchers study OR11L1 to understand fundamental olfactory signaling mechanisms, investigate potential non-olfactory functions of olfactory receptors in various tissues, and explore structure-function relationships in GPCR biology. The protein is primarily studied using antibody-based detection methods that allow researchers to visualize its expression patterns and localization within cells and tissues .

What types of OR11L1 antibodies are available for research applications?

Several types of OR11L1 antibodies have been developed for scientific applications, with most being rabbit-derived polyclonal antibodies targeting specific regions of the human OR11L1 protein. The available antibodies can be categorized based on their targeting epitopes and applications:

Most commercially available antibodies are unconjugated and have been validated for use with human samples . When selecting an OR11L1 antibody, researchers should consider which epitope is most appropriate for their specific application and experimental design.

What are the validated applications for OR11L1 antibodies?

OR11L1 antibodies have been validated for several experimental applications, making them versatile tools for protein detection and analysis:

Scientific validation data demonstrates successful detection of OR11L1 in specific cell lines, with Western blot analysis showing a band at the expected molecular weight of approximately 36kDa in LOVO cell lysates and clear immunofluorescence staining patterns in A549 cells . These applications allow researchers to investigate OR11L1 expression levels, subcellular localization, and potential interactions with other proteins.

What sample types have been validated with OR11L1 antibodies?

OR11L1 antibodies have been validated primarily for use with human samples. The specific sample types that have shown successful results include:

• Human cell lines:

  • A549 cells (human lung adenocarcinoma epithelial cell line) have been validated for immunofluorescence applications

  • LOVO cells (human colon adenocarcinoma cell line) have been validated for Western blot applications

• Sample preparation formats:

  • Cell lysates for Western blot analysis

  • Fixed and permeabilized cells for immunofluorescence

  • Protein solutions for ELISA

Researchers should note that most available OR11L1 antibodies are specifically designed for human samples, and cross-reactivity with other species is generally not well-documented in the available literature . When working with non-human samples, additional validation steps may be necessary to confirm antibody specificity.

How do different epitope targets affect OR11L1 antibody performance?

The epitope (target region) of an OR11L1 antibody significantly influences its performance across different applications:

For detection of total OR11L1 protein, antibodies targeting well-conserved internal regions (e.g., aa 201-250) are often preferred . For studying protein modifications, C-terminal targeted antibodies may be more appropriate if post-translational modifications occur at other regions. When investigating protein-protein interactions, researchers should consider whether the epitope might be masked by interacting partners in the native state.

Some suppliers offer antibodies targeting different regions of OR11L1, allowing researchers to select the most appropriate one for their specific application . Using multiple antibodies targeting different epitopes can provide more comprehensive and reliable results.

What are optimal validation methods for OR11L1 antibodies?

Comprehensive validation of OR11L1 antibodies should include multiple approaches to confirm specificity and reliability:

• Positive controls:

  • Cell lines with confirmed OR11L1 expression (e.g., LOVO cells for Western blot, A549 cells for immunofluorescence)

  • Recombinant OR11L1 protein (if available)

  • Tissue samples with known expression

• Negative controls:

  • Peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

  • Secondary antibody only controls

  • Isotype controls (matched rabbit IgG)

  • Knockdown/knockout validation where OR11L1 expression is reduced or eliminated

• Specificity tests:

  • Western blot should show a predominant band at ~36kDa (the expected molecular weight of OR11L1)

  • Immunofluorescence patterns should be consistent with expected subcellular localization

  • Multiple antibodies targeting different epitopes should show concordant results

Published validation data for some OR11L1 antibodies includes Western blot analysis of LOVO cell lysates and immunofluorescence of A549 cells, with appropriate negative controls demonstrating the abolishment of signal when the antibody is pre-incubated with the immunizing peptide . This type of comprehensive validation increases confidence in the specificity and reliability of results.

How can OR11L1 antibodies be used in multiplex immunofluorescence studies?

For multiplex immunofluorescence involving OR11L1 detection alongside other proteins, several considerations are important:

• Antibody selection strategies:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • For OR11L1, rabbit polyclonal antibodies are most common , so pair with mouse, goat, or chicken antibodies for other targets

  • If multiple rabbit antibodies must be used, consider directly conjugated antibodies or sequential staining protocols

• Secondary antibody options for OR11L1 (rabbit) primary antibodies:

  • Goat anti-rabbit IgG with various fluorophores (FITC, AP, Biotin, HRP)

  • Alpaca anti-rabbit IgG conjugates (Alexa Fluor 488, 568, 647)

  • Donkey anti-rabbit IgG FITC

• Multiplex protocol optimization:

  • Sequential staining may be necessary if antibodies are from the same species

  • Careful blocking between rounds to prevent cross-reactivity

  • Consider spectral unmixing for overlapping fluorophores

• Essential controls for multiplex experiments:

  • Single-stain controls for each antibody to confirm specificity and lack of bleed-through

  • Combined isotype controls to assess background

  • Unstained samples to evaluate autofluorescence

Several compatible secondary antibody options for rabbit anti-OR11L1 antibodies are available for multiplex applications, including those conjugated to various fluorophores suitable for multicolor imaging . Proper selection of antibody combinations and rigorous controls are essential for obtaining reliable results in multiplex studies.

What are the recommended protocols for Western blot using OR11L1 antibodies?

An optimized Western blot protocol for OR11L1 detection includes the following steps:

Sample preparation:

• Lyse cells (e.g., LOVO cells, which have shown positive results ) in RIPA buffer supplemented with protease inhibitors

• Quantify protein concentration using BCA or Bradford assay

• Prepare samples in Laemmli buffer with reducing agent

• Heat at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Load 20-50μg protein per lane

• Separate on 10-12% SDS-PAGE (OR11L1 molecular weight: ~36kDa)

• Transfer to PVDF or nitrocellulose membrane (0.45μm)

Antibody incubation:

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

• Incubate with primary anti-OR11L1 antibody at 1:500-1:1000 dilution in blocking buffer overnight at 4°C

• Wash 3x with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature

• Wash 3x with TBST, 5 minutes each

Detection:

• Apply ECL substrate and develop using film or digital imager

• Expected band: ~36kDa

Essential controls:

• Positive control: LOVO cell lysate

• Negative control: Primary antibody pre-incubated with immunizing peptide

• Loading control: β-actin or GAPDH

This protocol has been effectively used with anti-OR11L1 antibodies as demonstrated in published validation data showing specific detection in LOVO cell lysates .

How should immunofluorescence experiments with OR11L1 antibodies be optimized?

An optimized immunofluorescence protocol for OR11L1 detection includes:

Sample preparation:

• Culture cells on coverslips or chamber slides (A549 cells have shown positive results )

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

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

Staining procedure:

• Block with 5% normal serum in PBS for 1 hour

• Incubate with primary anti-OR11L1 antibody at 1:100-1:500 dilution in blocking buffer overnight at 4°C

• Wash 3x with PBS, 5 minutes each

• Incubate with fluorophore-conjugated secondary antibody at 1:500 dilution for 1 hour at room temperature in the dark

• Wash 3x with PBS, 5 minutes each

• Counterstain nuclei with DAPI (1:1000) for 5 minutes

• Mount with anti-fade mounting medium

Controls:

• Positive control: A549 cells

• Negative controls:

  • Primary antibody pre-incubated with immunizing peptide

  • Secondary antibody only

  • Isotype control (matching rabbit IgG)

Optimization parameters:

• Fixation: Try different fixatives if standard PFA doesn't work (methanol, acetone)

• Antibody concentration: Titrate to determine optimal dilution (typically 1:100-1:500)

• Antigen retrieval: Consider if signal is weak (citrate buffer, pH 6.0)

This protocol has been successfully used with anti-OR11L1 antibodies as shown in published validation images demonstrating specific staining in A549 cells .

What are the recommended dilutions for different OR11L1 antibody applications?

Based on validated protocols and manufacturer recommendations, the following dilutions are advised for OR11L1 antibodies in different applications:

For new applications or cell types, performing a titration experiment with a dilution series (e.g., 1:100, 1:200, 1:500, 1:1000) is recommended to identify the optimal concentration. Select the dilution that provides the strongest specific signal with minimal background. Different lots may require slight adjustments in dilution, and sample-specific factors like expression level should also be considered when determining the optimal dilution.

Most OR11L1 antibodies are supplied in liquid form with buffer containing glycerol and sodium azide, which helps maintain stability . Always follow manufacturer storage recommendations (typically -20°C with avoidance of freeze-thaw cycles) to maintain antibody activity.

Why might Western blot with OR11L1 antibodies show non-specific bands?

Non-specific bands in OR11L1 Western blots can result from several factors:

• Cross-reactivity with related proteins:

  • OR11L1 belongs to the olfactory receptor family, which has many members with sequence similarity

  • Solution: Use antibodies targeting unique regions of OR11L1 and validate with peptide competition assays

• Protein degradation products:

  • Degraded OR11L1 can appear as multiple lower molecular weight bands

  • Solution: Use fresh samples, add protease inhibitors during lysis, keep samples cold

• Insufficient blocking:

  • Inadequate blocking can lead to non-specific antibody binding

  • Solution: Increase blocking time (≥1 hour), try different blocking agents (milk vs. BSA)

• Secondary antibody issues:

  • Cross-reactivity of secondary antibody

  • Solution: Try alternative secondary antibodies, include additional blocking steps

Validation approaches to confirm specificity include peptide competition (pre-incubation with immunizing peptide should eliminate specific bands but not non-specific ones) , testing multiple antibodies targeting distinct epitopes of OR11L1, and using appropriate positive and negative controls.

Published validation data for OR11L1 antibodies shows a clean Western blot with a single band at the expected molecular weight (~36kDa) in LOVO cell lysates, with signal completely abolished by peptide competition , suggesting that with optimal conditions, specific detection is achievable.

How can background signal be reduced in OR11L1 immunofluorescence staining?

High background in OR11L1 immunofluorescence can be addressed through several optimization strategies:

• Non-specific antibody binding:

  • Increase blocking time (≥1 hour) and concentration (5-10% serum)

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

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

• Insufficient washing:

  • Increase number of washes (5-6 times instead of 3)

  • Extend washing time (10 minutes per wash)

  • Use gentle agitation during washing

• Antibody concentration too high:

  • Dilute primary antibody further (try 1:500 instead of 1:100)

  • Dilute secondary antibody (typically 1:500-1:1000)

• Autofluorescence:

  • Include Sudan Black B treatment (0.1% in 70% ethanol for 20 minutes)

  • Use commercial autofluorescence reducers

  • Change fluorophore to avoid spectral overlap with autofluorescence

A sequential immunostaining approach with double-blocking can significantly reduce background. Pre-adsorption of antibodies with cells not expressing the target can also help remove antibodies that bind non-specifically to other cellular components.

Published validation data for OR11L1 antibodies shows clean immunofluorescence staining in A549 cells with minimal background, which is completely eliminated by peptide competition , indicating that with proper optimization, specific detection with low background is achievable.

What should be done if OR11L1 antibodies fail to detect the target in validated samples?

When OR11L1 antibodies fail to generate expected signals in validated samples, a systematic troubleshooting approach should be followed:

• Verify antibody functionality:

  • Test antibody on positive control samples (e.g., LOVO or A549 cells)

  • Check antibody expiration date and storage conditions

  • Run a dot blot with the immunizing peptide to confirm antibody activity

• Sample-related factors:

  • Confirm OR11L1 expression in your sample (check mRNA levels by RT-PCR)

  • Ensure protein hasn't been degraded (use fresh samples, proper protease inhibitors)

  • Verify sample preparation preserves the epitope (try different lysis buffers)

• Application-specific optimizations:

  For Western blot:

  • Try different protein extraction methods (RIPA, NP-40, or urea-based buffers)

  • Optimize protein loading (increase amount to 50-100μg)

  • Try different transfer conditions (wet transfer instead of semi-dry)

  • Use enhanced chemiluminescence substrate with higher sensitivity

  For immunofluorescence:

  • Try different fixation methods (PFA, methanol, acetone)

  • Include antigen retrieval step (citrate buffer pH 6.0, heat-induced)

  • Increase antibody concentration and incubation time

  • Try different detection systems (amplification with tyramide signal amplification)

The validation data for OR11L1 antibodies confirms successful detection in specific cell lines (LOVO for WB, A549 for IF) , providing reference points for troubleshooting experiments.

How can the sensitivity of OR11L1 detection be improved in low-expression samples?

Enhancing detection sensitivity for OR11L1 in samples with low expression levels can be achieved through several approaches:

For Western blot sensitivity enhancement:

• Sample enrichment:

  • Increase protein loading (50-100μg per lane)

  • Concentrate samples using precipitation (TCA, acetone)

  • Perform subcellular fractionation if OR11L1 is enriched in specific compartments

• Detection system optimization:

  • Use high-sensitivity ECL substrates (femtogram detection range)

  • Consider fluorescent Western blot systems

  • Try biotin-streptavidin amplification systems

• Antibody conditions:

  • Reduce primary antibody dilution (1:250 instead of 1:500)

  • Extend primary antibody incubation (overnight at 4°C)

For immunofluorescence sensitivity enhancement:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) can increase sensitivity 10-100 fold

  • Use biotinylated secondary antibody followed by fluorescent streptavidin

• Protocol adjustments:

  • Increase primary antibody concentration (1:50 instead of 1:100)

  • Extend incubation time (48-72 hours at 4°C)

• Optical optimization:

  • Use confocal microscopy with increased laser power and detector gain

  • Apply deconvolution algorithms to enhance signal-to-noise ratio

By combining multiple sensitivity-enhancing approaches while maintaining appropriate controls to validate weak signals, detection of low-abundance OR11L1 can be significantly improved while preserving specificity.

What controls are essential when working with OR11L1 antibodies?

A comprehensive set of controls is critical for reliable OR11L1 antibody experiments:

• Positive controls:

  • Samples known to express OR11L1 (e.g., LOVO cells for WB, A549 cells for IF)

  • Recombinant OR11L1 protein (if available)

  • Overexpression systems (cells transfected with OR11L1 expression vector)

• Negative controls:

  • Peptide competition: Primary antibody pre-incubated with immunizing peptide

  • Isotype control: Matched rabbit IgG at equivalent concentration

  • Secondary antibody only (omit primary antibody)

• Application-specific controls:

  For Western blot:

  • Molecular weight markers to confirm band size (~36kDa for OR11L1)

  • Loading control (β-actin, GAPDH) to normalize protein amounts

  For immunofluorescence:

  • DAPI nuclear counterstain to assess cell morphology

  • Cytoskeletal or membrane markers to provide cellular context

  • Unstained sample to assess autofluorescence

  For ELISA:

  • Standard curve using recombinant protein (if available)

  • Blank wells (no sample) to establish baseline

Published validation data for OR11L1 antibodies demonstrates the use of both positive controls (LOVO and A549 cells) and peptide competition controls, showing complete elimination of specific signals when the antibody is blocked with the immunizing peptide . These controls are essential for distinguishing specific from non-specific signals and validating experimental results.