LDLRAD1 Antibody

What is LDLRAD1 and its relationship to the LDLR family?

LDLRAD1 (Low-density lipoprotein receptor class A domain-containing protein 1) belongs to the LDLR (Low-Density Lipoprotein Receptor) family . This protein contains LDL receptor class A domains that characterize this receptor family. LDLRAD1 is part of a broader network of receptors involved in lipoprotein metabolism and cellular signaling pathways. The LDLR family includes several structurally homologous receptors composed of modular structures such as the LDL receptor, VLDL receptor, apoE receptor 2, and others that play diverse roles in various biological processes . Understanding LDLRAD1's phylogenetic relationship to these receptors provides context for its potential functional significance in lipid metabolism and other cellular processes.

What are the structural characteristics of LDLRAD1 protein?

LDLRAD1 has a calculated molecular weight of approximately 22 kDa, though it typically appears at around 13 kDa in Western blots, suggesting possible post-translational modifications or alternative processing . The protein exists in at least three isoforms that can be recognized by certain antibodies . According to available product information, recombinant human LDLRAD1 protein (such as Abcam's ab127304) represents a fragment in the 97 to 198 amino acid range . The protein's structure includes LDL receptor class A domains, which are cysteine-rich regions that typically function in ligand binding within this protein family. These structural features are important considerations when designing experiments and selecting appropriate antibodies for detection.

What is currently known about LDLRAD1 tissue expression patterns?

Based on antibody validation data, LDLRAD1 expression has been detected in mouse brain and liver tissues . This expression pattern differs somewhat from the classical LDL receptor, which is highly expressed in the liver but shows a different distribution pattern. Unlike the extensively characterized LRP1 (LDL receptor-related protein 1), which has well-documented expression in the vasculature, central nervous system, macrophages, and adipocytes , the complete tissue distribution profile for LDLRAD1 requires further characterization through comprehensive immunohistochemistry or transcriptomic studies. Researchers investigating LDLRAD1 should consider these known expression sites when designing experiments and selecting appropriate control tissues.

What types of LDLRAD1 antibodies are commercially available for research?

Several types of LDLRAD1 antibodies are available for research applications:

Most available antibodies are rabbit polyclonal antibodies raised against synthetic peptides derived from human LDLRAD1 . These antibodies are typically available in unconjugated form, though some suppliers offer custom conjugation services with various fluorophores and enzymes for specialized applications . When selecting an antibody, researchers should consider the intended application, species reactivity, and validation data available.

What validation steps should be performed before using a new LDLRAD1 antibody?

Thorough validation of LDLRAD1 antibodies should include:

• Positive control testing using tissues known to express LDLRAD1 (mouse brain and liver have been reported as positives)

• Negative controls (tissues or cell lines with low/no expression)

• Western blot analysis to confirm the band appears at the expected molecular weight (~13 kDa observed, though calculated at 22 kDa)

• Evaluation of cross-reactivity with related LDLR family proteins

• If possible, additional validation using knockout or knockdown models

• Titration experiments to determine optimal working concentrations for specific applications

Researchers should also verify that the antibody recognizes the specific epitope or region of interest, especially when studying particular domains or isoforms of LDLRAD1. This validation is critical for ensuring the reliability and reproducibility of experimental results.

How do LDLRAD1 antibodies compare to antibodies against other LDLR family members?

When comparing LDLRAD1 antibodies to those targeting other LDLR family members (such as LDLR itself):

• Specificity considerations: LDLR family members share structural similarities, particularly in their class A domains. Researchers must verify that LDLRAD1 antibodies do not cross-react with other family members .

• Applications range: LDLR antibodies are extensively validated for multiple applications including Western blot, immunoprecipitation, immunohistochemistry, and flow cytometry . In contrast, LDLRAD1 antibodies typically have more limited validation, primarily for Western blot and ELISA .

• Species reactivity: LDLR antibodies often have broader cross-species reactivity profiles, with products available for human, mouse, rat, and other species . LDLRAD1 antibodies generally have more restricted species reactivity, with most validated only for human and sometimes mouse samples .

• Validation depth: Due to greater research focus on LDLR, antibodies against this protein typically have more extensive validation data, including knockout controls and citations in published literature . Researchers working with LDLRAD1 antibodies should perform additional validation to ensure comparable reliability.

What are the optimal protocols for Western blot detection of LDLRAD1?

For optimal Western blot detection of LDLRAD1:

• Sample preparation:

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors

  • Include phosphatase inhibitors if investigating potential post-translational modifications

  • For membrane proteins like LDLRAD1, ensure thorough homogenization

• Gel electrophoresis:

  • Use 12-15% gels to properly resolve the 13 kDa observed molecular weight

  • Consider gradient gels if comparing LDLRAD1 with other LDLR family members

• Antibody dilutions:

  • Primary antibody: 1:200-1:1000 for Proteintech's antibody ; 1:500-1:3000 for other suppliers

  • Secondary antibody: Typically 1:5000-1:10000 HRP-conjugated anti-rabbit IgG

• Controls:

  • Include mouse brain or liver lysate as positive controls

  • Consider using recombinant LDLRAD1 protein as a reference standard

• Detection:

  • Enhanced chemiluminescence (ECL) is generally sufficient

  • For weak signals, consider more sensitive ECL substrates or longer exposure times

Optimization of these parameters for your specific experimental system is essential for reliable results.

How should immunohistochemistry experiments with LDLRAD1 antibodies be designed?

For successful immunohistochemistry (IHC) or immunofluorescence (IF) experiments with LDLRAD1 antibodies:

• Tissue preparation:

  • Use freshly collected or properly fixed tissue samples

  • For paraffin-embedded sections, consider antigen retrieval methods to expose epitopes

  • For frozen sections, optimize fixation time to balance epitope preservation and tissue morphology

• Antibody selection and validation:

  • Verify that your antibody is validated for IHC/IF applications

  • Test multiple antibody concentrations (typically starting at 1:100-1:500)

  • Include known positive tissue controls (brain and liver based on validation data)

• Protocol optimization:

  • Test different blocking solutions to minimize background (5-10% normal serum from the species of secondary antibody)

  • For membrane proteins like LDLRAD1, permeabilization conditions are critical

  • Optimize primary antibody incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature)

• Signal detection:

  • For chromogenic detection, optimize DAB development time

  • For fluorescence, select fluorophores with spectral properties compatible with other markers in multiplexed experiments

  • Include DAPI or similar nuclear counterstain for cell localization

• Controls:

  • Include no-primary-antibody controls to assess secondary antibody specificity

  • Use tissue from LDLRAD1-deficient animals or cells if available

What are the considerations for studying LDLRAD1 interactions with other proteins?

When investigating protein-protein interactions involving LDLRAD1:

• Co-immunoprecipitation (Co-IP) approaches:

  • Verify that your LDLRAD1 antibody is suitable for immunoprecipitation

  • Choose lysis conditions that preserve native protein conformations

  • Consider crosslinking for transient or weak interactions

  • Use stringent controls including IgG control and reverse Co-IP

• Proximity-based methods:

  • Proximity ligation assays (PLA) can detect interactions with spatial resolution

  • FRET or BRET approaches require tagged versions of LDLRAD1 and potential partners

  • For these techniques, validate that tags do not interfere with normal protein localization or function

• Protein domain considerations:

  • LDL receptor family members like LDLRAD1 interact with proteins through specific domains

  • Consider domain-specific antibodies or constructs to map interaction regions

  • The intracellular domain of LDLR family members often contains interaction motifs for cytoplasmic adaptor proteins

• Stimulus-dependent interactions:

  • Some LDLR family protein interactions are regulated by phosphorylation or other modifications

  • Design experiments to capture different cellular states (resting vs. stimulated)

• Control experiments:

  • Include structurally related proteins (other LDLR family members) as specificity controls

  • Consider using recombinant protein domains to compete for interactions

Why does LDLRAD1 show a discrepancy between calculated and observed molecular weights?

The discrepancy between LDLRAD1's calculated molecular weight (22 kDa) and its observed Western blot mobility (13 kDa) could be attributed to several factors:

• Post-translational modifications:

  • Proteolytic processing may generate smaller fragments

  • Glycosylation or other modifications can affect gel mobility

  • The LDL receptor family members undergo various post-translational modifications

• Protein structure influences:

  • High content of charged residues can affect SDS binding and migration

  • Incomplete denaturation of certain domains can cause anomalous migration

  • Membrane proteins often exhibit unexpected migration patterns

• Isoform detection:

  • LDLRAD1 has multiple isoforms that may differ in size

  • The antibody epitope location determines which isoforms or fragments are detected

When interpreting Western blot results, researchers should consider:

• Running recombinant LDLRAD1 protein as a size reference

• Using multiple antibodies targeting different epitopes

• Complementing protein analysis with mRNA studies to identify expressed isoforms

What are common technical challenges when detecting LDLRAD1 and how can they be addressed?

When troubleshooting, a systematic approach that changes one variable at a time will help identify the source of technical problems. Document all experimental conditions carefully to ensure reproducibility once optimal conditions are established.

How should researchers interpret LDLRAD1 expression patterns in relation to other LDLR family members?

When analyzing LDLRAD1 expression patterns:

• Comparative expression analysis:

  • LDLR family members show tissue-specific expression patterns

  • Compare LDLRAD1 expression with other family members in the same experimental system

  • Consider using a panel of antibodies against multiple LDLR family proteins

• Functional context interpretation:

  • LRP1, another family member, has important functions in the vasculature, CNS, macrophages, and adipocytes

  • Evaluate LDLRAD1 expression in these contexts to understand potential functional overlap or divergence

  • Consider the specific cellular compartments where LDLRAD1 is detected compared to other family members

• Regulatory considerations:

  • LDLR family member expression can be regulated by metabolic conditions

  • Assess whether LDLRAD1 shows similar regulatory patterns to LDLR or distinct regulation

• Developmental and pathological contexts:

  • Compare expression patterns across normal development and in disease states

  • Determine if LDLRAD1 expression correlates with or diverges from other family members

• Data integration:

  • Combine protein detection data with transcriptomic and proteomic datasets

  • Use publicly available datasets to complement experimental findings

  • Consider single-cell approaches to resolve cell-specific expression patterns

How can LDLRAD1 antibodies be utilized in studying receptor trafficking and endocytosis?

LDLRAD1 antibodies can be valuable tools for investigating receptor trafficking:

• Internalization assays:

  • Surface-bound antibodies can be used to track receptor internalization over time

  • Live-cell imaging with fluorescently labeled antibodies can visualize trafficking dynamics

  • Flow cytometry can quantify surface vs. internalized receptors

• Co-trafficking studies:

  • The LDL receptor family utilizes specialized trafficking machinery including RAP (receptor-associated protein)

  • Examine whether LDLRAD1 utilizes similar chaperones and trafficking pathways

  • Investigate co-localization with markers of endocytic compartments

• Recycling vs. degradation fate:

  • Pulse-chase experiments with antibodies can determine receptor half-life and recycling efficiency

  • Examine co-localization with lysosomal markers to assess degradation patterns

• Mutational analysis:

  • Antibodies recognizing specific domains can help determine which regions are critical for trafficking

  • Compare trafficking of LDLRAD1 with other family members that contain similar motifs

• Regulation studies:

  • Investigate how various stimuli affect LDLRAD1 trafficking compared to canonical LDLR

  • Examine potential roles of post-translational modifications in trafficking regulation

What approaches can be used to investigate LDLRAD1 in pathological conditions?

For studying LDLRAD1 in disease contexts:

• Expression analysis in disease tissues:

  • Examine LDLRAD1 levels in relevant pathological samples

  • Compare with other LDLR family members that have established roles in diseases

• Functional studies in disease models:

  • Overexpression or knockdown of LDLRAD1 in cellular or animal models

  • Assess effects on disease-relevant endpoints

  • LDLR family members have roles in cardiovascular disease and neurological disorders

• Genetic association studies:

  • Evaluate potential correlations between LDLRAD1 genetic variants and disease susceptibility

  • Compare with known pathogenic variants in other LDLR family genes

• Therapeutic targeting assessment:

  • Evaluate antibodies as potential blocking agents for LDLRAD1 function

  • Determine epitopes that might be relevant for therapeutic intervention

• Biomarker potential:

  • Investigate whether soluble forms of LDLRAD1 exist in biological fluids

  • Develop quantitative assays using antibody pairs for ELISA detection

How can researchers differentiate between various LDLRAD1 isoforms in experimental systems?

Distinguishing between LDLRAD1 isoforms requires specialized approaches:

• Isoform-specific detection strategies:

  • Use antibodies targeting unique regions present in specific isoforms

  • Design isoform-specific PCR primers for transcript analysis

  • Consider mass spectrometry approaches for definitive isoform identification

• Expression system utilization:

  • Create expression constructs for individual isoforms as reference standards

  • Use these in side-by-side comparisons with endogenous protein

• High-resolution protein separation:

  • Utilize gradient gels or Phos-tag gels for improved separation of closely sized isoforms

  • Consider 2D gel electrophoresis to separate based on both size and charge

• Functional characterization:

  • Assess whether different isoforms show distinct subcellular localization

  • Evaluate functional differences in binding assays or trafficking studies

  • Determine if isoforms are differentially regulated in specific contexts

• Knockout/knockin approaches:

  • Generate isoform-specific knockout models if technically feasible

  • Use gene editing to tag specific isoforms for differential detection

By implementing these advanced approaches, researchers can gain deeper insights into the specific roles of LDLRAD1 isoforms in both normal physiology and disease states.