LAMP2 Recombinant Monoclonal Antibody

What is LAMP2 and why is it important in cellular research?

LAMP2 (also known as CD107b) is a transmembrane glycoprotein that serves as a major component of lysosomal membranes. Mature human LAMP2 consists of a 347 amino acid intralumenal domain, a 24 amino acid transmembrane segment, and a 35 amino acid cytoplasmic tail . The protein plays critical roles in multiple cellular processes including:

• Lysosomal biogenesis and pH regulation

• Chaperone-mediated autophagy

• Autophagosome-lysosome fusion

• Protein degradation pathways

• Antigen presentation in immune cells

LAMP2 functions as a direct inhibitor of the proton channel TMEM175, facilitating lysosomal acidification required for optimal hydrolase activity . Its importance extends beyond basic lysosomal function, as it also participates in immune cell activation and exosome biology . Mutations in LAMP2 cause Danon disease, characterized by cardiomyopathy, myopathy, and cognitive impairment, highlighting its physiological significance.

What are the structural characteristics and isoforms of LAMP2?

LAMP2 is an approximately 110 kDa transmembrane glycoprotein with several distinctive structural features:

• The lumenal domain is organized into two heavily N-glycosylated regions separated by a Ser/Pro-rich linker carrying minor O-linked glycosylation

• While the calculated molecular weight is approximately 45 kDa, the mature protein typically appears at 100-130 kDa on Western blots due to extensive glycosylation

• The protein contains multiple disulfide bonds that maintain its tertiary structure

Alternative splicing generates multiple LAMP2 isoforms with substituted juxtamembrane lumenal regions, transmembrane segments, and cytoplasmic tails . The most well-characterized isoforms include:

• LAMP2A: Critical for chaperone-mediated autophagy, functioning as a receptor for substrate proteins

• LAMP2B: Predominantly expressed in cardiac and skeletal muscle

• LAMP2C: Involved in RNA and DNA autophagy

Each isoform has unique C-terminal sequences that determine their specific functions and interactions within cellular pathways.

What types of LAMP2 recombinant monoclonal antibodies are available for research?

Several types of LAMP2 recombinant monoclonal antibodies are commercially available, differentiated by host species, clones, and target epitopes:

Many of these antibodies have been extensively validated and cited in numerous publications, with some clones like GL2A7 cited in over 260 scientific papers . When selecting an antibody, researchers should consider the target species compatibility, intended applications, and whether isoform specificity is required for their particular research question.

How do I determine which LAMP2 antibody is suitable for my specific application?

Selecting the appropriate LAMP2 antibody requires consideration of several key factors:

• Target species compatibility: Confirm cross-reactivity with your species of interest. Most LAMP2 antibodies work well with human samples, but murine cross-reactivity varies by clone .

• Application suitability: Different clones perform optimally in different applications. For example:

  • For Western blotting: Clones H4B4 and ARC54762 are highly recommended with dilutions of 1:10,000-1:160,000

  • For immunohistochemistry: GL2A7 and ARC54762 perform well at 1:200-1:2,000 dilutions

  • For flow cytometry: Clone 743320 has been validated for detecting LAMP2 in fixed and permeabilized cells

• Epitope recognition: Consider whether the antibody recognizes:

  • All LAMP2 isoforms (most commercial antibodies)

  • Specific isoforms (required for specialized studies of LAMP2A, B, or C functions)

  • Conformational vs. linear epitopes (affects performance in denatured vs. native conditions)

• Validation evidence: Review manufacturer data showing antibody specificity through:

  • Knockout/knockdown controls

  • Peptide competition assays

  • Detection of expected molecular weight bands

  • Performance in multiple cell lines or tissues

The most reliable approach is to test multiple antibodies in preliminary experiments with appropriate positive and negative controls relevant to your experimental system.

What are the optimal protocols for detecting LAMP2 by Western blotting?

Detecting LAMP2 by Western blotting requires specific considerations due to its heavily glycosylated nature and membrane localization:

• Sample preparation:

  • Use RIPA or NP-40 buffer supplemented with protease inhibitors

  • Include 1-2% SDS for complete solubilization of membrane proteins

  • Add phosphatase inhibitors if phosphorylation status is important

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Protein loading and separation:

  • Load 25-50 μg of total protein per lane

  • Use 7.5-10% polyacrylamide gels to properly resolve the 100-130 kDa glycosylated form

  • Include positive control lysates (HeLa, U937, or Jurkat cells)

• Transfer conditions:

  • Employ wet transfer rather than semi-dry for complete transfer of large proteins

  • Use PVDF membranes (0.45 μm pore size) for better protein retention

  • Transfer at lower voltage (30V) overnight at 4°C for improved efficiency

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or 3-5% BSA in TBST

  • Dilute primary antibodies appropriately (1:40,000-1:160,000 for high-affinity clones)

  • Incubate with primary antibody overnight at 4°C for optimal sensitivity

  • Wash extensively (5 × 5 minutes) before secondary antibody incubation

• Detection considerations:

  • Use HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • Expect bands at 100-130 kDa due to glycosylation (calculated MW is ~45 kDa)

  • For deglycosylation studies, treat samples with PNGase F before electrophoresis

This protocol has been validated with multiple LAMP2 antibodies including ARC54762, H4B4, and GL2A7 across various cell types .

How should I optimize LAMP2 detection by immunofluorescence microscopy?

For successful immunofluorescence detection of LAMP2:

• Cell preparation and fixation:

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

  • Fix with 4% paraformaldehyde for 15-20 minutes at room temperature

  • For optimal lysosomal preservation, avoid methanol fixation which can distort vesicular structures

• Permeabilization options:

  • For total LAMP2: Use 0.1-0.5% saponin (preferred for lysosomal proteins over Triton X-100)

  • For surface LAMP2 only: Omit permeabilization step

• Blocking and antibody incubation:

  • Block with 5-10% normal serum (matching secondary antibody species) plus 1% BSA

  • Dilute primary LAMP2 antibody appropriately (1:200-1:1,000 for most clones)

  • Incubate overnight at 4°C in a humidified chamber

  • Wash thoroughly (3-5 × 5 minutes) with PBS containing 0.025% saponin

• Detection and counterstaining:

  • Use fluorophore-conjugated secondary antibodies at manufacturer-recommended dilutions

  • Include DAPI or Hoechst for nuclear counterstaining

  • Mount with anti-fade medium containing glycerol or similar agents

• Imaging considerations:

  • Expect punctate cytoplasmic staining pattern consistent with lysosomal localization

  • Use confocal microscopy for optimal resolution of lysosomal structures

  • Consider co-staining with other lysosomal markers (e.g., LAMP1) for validation

For co-localization studies, LAMP2 antibodies from rat (GL2A7) can be combined with antibodies from different species targeting other proteins of interest . Z-stack acquisition is recommended for comprehensive analysis of lysosomal distribution throughout the cell volume.

What protocol should I follow for LAMP2 detection by flow cytometry?

Flow cytometric analysis of LAMP2 requires consideration of whether total or surface expression is being measured:

• Cell preparation:

  • Harvest cells in exponential growth phase

  • Prepare single-cell suspensions (1-5 × 10^6 cells/ml)

  • Wash in PBS containing 2% FBS (flow buffer)

• For total LAMP2 detection (most common):

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1% saponin in flow buffer (maintain saponin in all subsequent steps)

  • Block with 5% normal serum from secondary antibody species

• For surface LAMP2 detection (activation marker):

  • Use unfixed cells or gentle fixation (1% paraformaldehyde, 5 minutes)

  • Omit permeabilization step

  • Keep cells cold (4°C) throughout the procedure

• Antibody staining:

  • Use approximately 0.1 μg antibody per 10^6 cells (clone-dependent)

  • Incubate for 30-60 minutes at 4°C

  • Wash 3× in appropriate buffer (with or without saponin)

  • Apply fluorophore-conjugated secondary antibody if using unconjugated primary

• Controls and analysis:

  • Include isotype control matched to primary antibody concentration

  • Prepare single-color controls for compensation if performing multicolor analysis

  • Gate on live cells using viability dye if analyzing fixed/permeabilized samples

  • Analyze LAMP2 as median fluorescence intensity rather than percent positive

This protocol has been validated using various LAMP2 antibodies including clone 743320 and H4B4 . For detection of LAMP2 in HeLa cells, paraformaldehyde fixation followed by saponin permeabilization has shown robust results with minimal background .

How can LAMP2 antibodies be used to study chaperone-mediated autophagy?

Chaperone-mediated autophagy (CMA) is a selective form of autophagy where LAMP2A serves as the lysosomal receptor. LAMP2 antibodies can be instrumental in studying this process:

• Isoform-specific detection:

  • If using general LAMP2 antibodies, complement with LAMP2A-specific antibodies when available

  • Monitor LAMP2A multimerization (the active form for CMA) via non-reducing gels

  • Track changes in LAMP2A levels during starvation or stress conditions

• Co-immunoprecipitation studies:

  • Use LAMP2 antibodies to immunoprecipitate protein complexes

  • Probe for CMA substrate proteins (e.g., GAPDH, NLRP3, MLLT11)

  • Detect chaperones like HSPA8/HSC70 that interact with LAMP2 during CMA

• Microscopy-based approaches:

  • Perform immunofluorescence to visualize LAMP2 and CMA substrates

  • Analyze colocalization of substrates with LAMP2-positive lysosomes

  • Conduct live-cell imaging with fluorescently tagged LAMP2 and substrate proteins

• Functional CMA assays:

  • Use LAMP2 antibodies to monitor changes in CMA activity under various conditions

  • Combine with lysosomal isolation techniques to study direct protein translocation

  • Assess the impact of manipulating LAMP2 expression on degradation of known CMA substrates

LAMP2 functions by binding target proteins and targeting them for lysosomal degradation . In this process, it acts downstream of chaperones like HSPA8/HSC70, which recognize and bind substrate proteins and mediate their recruitment to lysosomes . Quantitative analysis of LAMP2-substrate interactions provides valuable insights into CMA regulation in various physiological and pathological contexts.

How can LAMP2 antibodies be utilized to study antigen presentation pathways?

LAMP2 has been identified as an endocytic receptor on human dendritic cells that routes cargo into immunogenic exosomes. LAMP2 antibodies can be used to study this pathway:

• Tracking receptor-mediated antigen uptake:

  • Conjugate antigens directly to anti-LAMP2 antibodies (e.g., clone H4B4)

  • Compare with other targeting strategies (e.g., DEC-205, DC-SIGN)

  • Monitor internalization kinetics and subcellular trafficking

• Analysis of antigen routing:

  • Use immunofluorescence microscopy to track LAMP2-bound antigens

  • Determine colocalization with HLA-DR in MIIC compartments

  • Compare surface presentation versus exosomal loading

• Exosome characterization:

  • Isolate exosomes from dendritic cells treated with LAMP2-targeted antigens

  • Quantify antigenic content in exosomes versus cell-surface presentation

  • Assess T cell stimulatory capacity of these exosomes

Research has shown that surface LAMP-2 is rapidly internalized upon ligation and traffics to the MIIC compartment, similar to known DC endocytic receptors . Surprisingly, despite this trafficking pattern, monocyte-derived dendritic cells pulsed with antigen conjugated to anti-LAMP-2 antibodies express fewer antigen-derived peptides in the HLA class II peptidome and evoke less T cell proliferation . Instead, antigens are selectively routed into highly immunogenic exosomes that stimulate robust CD4 T cell responses.

This pathway represents a novel mechanism for generating immunogenic extracellular vesicles that may contribute to immune responses both locally and at distant sites.

What methodologies can be used to study LAMP2's role in autophagosome-lysosome fusion?

LAMP2 plays a critical role in autophagosome-lysosome fusion, and studying this function requires specific methodological approaches:

• Colocalization analysis:

  • Use LAMP2 antibodies to mark lysosomes in fixed cells

  • Co-stain with autophagosome markers (LC3-II, ATG16L1)

  • Quantify colocalization under various conditions (starvation, drug treatments)

  • Employ super-resolution microscopy for detailed analysis of fusion sites

• Molecular mechanism investigation:

  • Examine STX17 accumulation on autophagosomes in LAMP2-deficient cells

  • Analyze VAMP8 and other SNARE proteins in relation to LAMP2

  • Study the impact of LAMP2 mutations on fusion efficiency

• Functional autophagy assays:

  • Compare autophagy flux in LAMP2-depleted versus control cells

  • Measure LC3-II accumulation with/without lysosomal inhibitors

  • Assess degradation of long-lived proteins or specific autophagy substrates

• Live-cell imaging approaches:

  • Use fluorescently-tagged LAMP2 constructs with autophagosomal markers

  • Monitor fusion events in real-time

  • Calculate fusion rates under various experimental conditions

Research has revealed that cells lacking LAMP2 express normal levels of VAMP8 but fail to accumulate STX17 on autophagosomes, which is likely the explanation for the defective fusion between autophagosomes and lysosomes in these cells . This finding highlights LAMP2's role in recruiting or stabilizing key fusion machinery components, providing mechanistic insight into its function in autophagy.

What are common issues when detecting LAMP2 by Western blotting and how can they be resolved?

Western blotting for LAMP2 can present several challenges due to its extensive glycosylation and membrane localization:

For optimal results, include validated positive control lysates such as HeLa, U937, or Jurkat cells, which are known to express detectable levels of LAMP2 . Consider non-reducing conditions for some antibodies, as certain epitopes may be conformation-dependent.

What controls should I include when using LAMP2 antibodies?

Proper controls are essential for generating reliable and interpretable results with LAMP2 antibodies:

• Positive controls:

  • Cell lines known to express LAMP2 (HeLa, U937, Jurkat cells)

  • Human liver tissue sections for IHC applications

  • Recombinant LAMP2 protein (for antibody validation)

• Negative controls:

  • Isotype control antibody matched to primary antibody class and concentration

  • Secondary antibody-only control to assess non-specific binding

  • LAMP2-deficient or knockdown samples when available

• Specificity controls:

  • Peptide competition assay (pre-incubation of antibody with immunizing peptide)

  • Validation with multiple antibodies targeting different LAMP2 epitopes

  • Deglycosylation controls to confirm identity of glycosylated bands

• Application-specific controls:

  • For immunofluorescence: Co-staining with other lysosomal markers

  • For flow cytometry: Fluorescence minus one (FMO) controls

  • For Western blotting: Loading controls and molecular weight markers

• Biological validation:

  • LAMP2 upregulation during starvation (autophagic response)

  • Surface LAMP2 increase upon activation of immune cells

  • Altered LAMP2 distribution after treatment with lysosomal inhibitors

Including these controls enables confident interpretation of results and troubleshooting of potential issues across different experimental platforms.

How can I distinguish between total LAMP2 and surface LAMP2 expression?

Distinguishing between total LAMP2 (predominantly lysosomal) and surface LAMP2 requires specific methodological approaches:

• Flow cytometry approach:

  • Total LAMP2: Fix cells with paraformaldehyde and permeabilize with saponin or similar agent

  • Surface LAMP2: Stain unfixed cells at 4°C, or use very mild fixation without permeabilization

  • Compare median fluorescence intensity between permeabilized and non-permeabilized samples

  • Surface LAMP2 serves as an activation marker for certain cell types (T cells, mast cells, monocytes, platelets)

• Microscopy-based distinction:

  • Total LAMP2: Standard fixation and permeabilization showing punctate cytoplasmic pattern

  • Surface LAMP2: Surface staining protocol showing membrane localization

  • Use confocal microscopy for accurate discrimination between surface and intracellular signals

  • Z-stack analysis to confirm genuine surface vs. intracellular localization

• Biochemical separation:

  • Surface biotinylation followed by streptavidin pull-down to isolate surface proteins

  • Probe for LAMP2 in biotinylated (surface) and non-biotinylated (internal) fractions

  • Compare with total cell lysate to determine relative distribution

• Kinetic analysis:

  • Monitor LAMP2 internalization from surface over time using antibody feeding assays

  • Track antibody-bound surface LAMP2 as it traffics to internal compartments

  • Quantify internalization rates under various experimental conditions

How are LAMP2 antibodies being used in neurodegenerative disease research?

LAMP2 antibodies are increasingly employed in neurodegenerative disease research to investigate lysosomal and autophagic dysfunction:

• Alzheimer's disease studies:

  • Quantifying LAMP2 expression in brain tissue from patients

  • Examining colocalization of amyloid-β and tau with LAMP2-positive lysosomes

  • Investigating impaired lysosomal clearance mechanisms

• Parkinson's disease applications:

  • Studying α-synuclein processing in LAMP2-positive compartments

  • Analyzing chaperone-mediated autophagy defects in dopaminergic neurons

  • Investigating LAMP2A expression in relation to disease progression

• Lysosomal storage disorders:

  • Danon disease (caused by LAMP2 mutations) research

  • Using LAMP2 as a marker for lysosomal accumulation in various storage disorders

  • Tracking therapeutic efficacy of treatments targeting lysosomal function

• Therapeutic development:

  • Screening compounds that enhance LAMP2 expression or function

  • Monitoring autophagy modulation using LAMP2 as a readout

  • Developing strategies to upregulate specific LAMP2 isoforms

LAMP2 plays a vital role in chaperone-mediated autophagy, which is increasingly recognized as dysfunctional in various neurodegenerative conditions . Methodological approaches include immunohistochemistry of post-mortem brain tissue, primary neuronal cultures, patient-derived iPSCs, and animal models of neurodegeneration.

How can LAMP2 antibodies be used to study exosome biology?

LAMP2 antibodies provide valuable tools for investigating exosome biology, particularly in immune contexts:

• Exosome characterization:

  • Western blotting for LAMP2 as an exosomal marker

  • Immunogold labeling with LAMP2 antibodies for electron microscopy

  • Flow cytometric analysis of LAMP2-positive extracellular vesicles

• Selective exosome isolation:

  • Immunoaffinity capture using anti-LAMP2 antibodies

  • Characterizing LAMP2-positive vs. LAMP2-negative exosome populations

  • Correlating LAMP2 content with exosome function

• Antigen loading in exosomes:

  • LAMP2-mediated routing of antigens into exosomes in dendritic cells

  • Quantitative assessment of antigen enrichment in exosomal fractions

  • Functional analysis of LAMP2-rich exosomes in immune responses

Research has shown that KLH-pulsed monocyte-derived dendritic cells produce exosomes containing significantly more HLA-DR and KLH than control exosomes, and these exosomes are uniquely effective sources of antigen in T cell proliferation assays . This represents a novel pathway where LAMP2 directs antigens away from surface presentation and into highly immunogenic extracellular vesicles.

What is the significance of anti-LAMP2 autoantibodies in autoimmune diseases?

Anti-LAMP2 autoantibodies have been identified in certain autoimmune conditions, though their prevalence and pathogenic role remain subjects of ongoing research:

• ANCA-associated vasculitis connection:

  • Some studies reported that >90% of patients with active pauci-immune glomerulonephritis had circulating anti-LAMP2 autoantibodies, though other research found lower frequencies

  • Many patients with anti-LAMP2 autoantibodies also have MPO-ANCA and PR3-ANCA

  • Various detection methods (ELISA, Western blot, immunofluorescence) show differing rates of positivity

• Molecular mimicry hypothesis:

  • Anti-LAMP2 antibodies may recognize a nine-amino-acid peptide in bacterial adhesion protein (FimH) from fimbriated gram-negative bacteria, including Escherichia coli

  • Immunological response triggered by bacterial infection might lead to production of autoantibodies to human LAMP2

  • Animal studies showed that rats immunized with FimH peptide developed pauci-immune glomerulonephritis and antibodies to human LAMP2

• Pathogenic potential:

  • Anti-LAMP2 autoantibodies have been proposed to be pathogenic in glomerulonephritis

  • They may disrupt lysosomal function and autophagy processes

  • If bacterial triggers are confirmed, this could have significant therapeutic implications

• Diagnostic considerations:

  • Healthy individuals with active urinary tract infections may have antibodies reactive against recombinant LAMP2 at frequencies similar to ANCA-positive patients

  • Various assay systems show differing sensitivities and specificities

  • Multiple testing methodologies may be required for accurate assessment

The study of anti-LAMP2 autoantibodies represents an emerging field with potential implications for understanding the pathogenesis and treatment of certain autoimmune conditions.