LAMC2 Antibody, FITC conjugated

What is LAMC2 and why is it important in research?

LAMC2 is an extracellular matrix protein that functions as the gamma-2 chain of laminin. It mediates cell attachment, migration, and organization of tissues during embryonic development through interactions with other extracellular matrix components . The significance of LAMC2 in research stems from its involvement in critical cellular processes including:

• Regulation of cell adhesion and migration

• Tissue formation and regeneration

• Association with signaling pathways (PI3K/AKT/mTOR)

• Role in tumor development and progression

LAMC2 has been identified as highly expressed in various cancers, making it an important subject for oncology research and a potential therapeutic target .

What are the specifications of commercially available LAMC2 Antibody, FITC Conjugated?

Commercially available LAMC2 Antibody, FITC Conjugated typically presents with the following specifications:

• Type: Primary Antibody

• Clonality: Polyclonal

• Host: Rabbit

• Reactivity: Human

• Isotype: IgG

• Label: FITC (Fluorescein isothiocyanate)

• Immunogen: Recombinant Human Laminin subunit gamma-2 protein (417-588AA)

• Purification Method: Protein G purified

These antibodies are generally developed for research applications and are not intended for diagnostic or therapeutic use.

What applications are suitable for LAMC2 Antibody, FITC Conjugated?

LAMC2 Antibody, FITC Conjugated is primarily designed for the following applications:

• ELISA: Recommended dilutions typically range from 1:100 to 1:500

• Immunofluorescence Microscopy: The FITC conjugation enables direct visualization of LAMC2 localization in fixed cells or tissue sections without secondary antibody requirements

• Flow Cytometry: For detection and quantification of LAMC2-expressing cells

• Live Cell Imaging: For studying real-time localization and dynamics of LAMC2, particularly useful in studies examining ER-mitochondria interactions

The pre-conjugated format eliminates the need for secondary antibody incubation steps, simplifying protocols and reducing background signals in fluorescence-based experiments.

How should LAMC2 Antibody, FITC Conjugated be stored and handled?

For optimal performance and longevity of LAMC2 Antibody, FITC Conjugated:

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

• Avoid repeated freeze-thaw cycles by aliquoting before freezing

• Protect from light to prevent photobleaching of the FITC fluorophore

• When working with the antibody, minimize exposure to light and maintain cold conditions when possible

• Follow manufacturer's recommendations for reconstitution if provided in lyophilized form

• Use aseptic techniques to prevent contamination

Proper storage and handling ensure maintenance of antibody binding capacity and fluorescence signal intensity over time.

How can LAMC2 Antibody be used to investigate ER-mitochondria interactions in cancer cells?

Recent research has demonstrated that LAMC2 participates in enhancing ER-mitochondria interactions and alleviating ER stress in cancer cells . To investigate this mechanism:

• Co-localization Studies: Use LAMC2 Antibody, FITC Conjugated alongside ER and mitochondrial markers (different fluorophores) to visualize triple co-localization at contact sites.

• Live Cell Imaging Protocol:

  • Transfect cells with mitochondrial marker (e.g., MitoTracker Red) and ER marker (e.g., ER-Tracker Blue)

  • Apply LAMC2 Antibody, FITC Conjugated using cell-penetrating peptide delivery systems for live imaging

  • Capture z-stack images using confocal microscopy

  • Perform quantitative analysis of co-localization coefficients using imaging software

• Proximity Ligation Assay (PLA):

  • Use LAMC2 Antibody in combination with antibodies against known ER-mitochondria tethering proteins

  • Quantify interaction events at subcellular resolution

Research has shown that LAMC2 co-localizes with both ER and mitochondria at their junction points, suggesting a role in facilitating communication between these organelles. This function appears critical for cancer cell adaptation to stress conditions .

What is the relationship between LAMC2 and EGFR signaling, and how can antibodies be used to study this interaction?

LAMC2 has been identified as a regulator of EGFR protein expression and stability in lung cancer . To study this relationship:

• Co-immunoprecipitation (Co-IP) Protocol:

  • Lyse cells in non-denaturing buffer

  • Pre-clear lysate with protein G beads

  • Incubate cleared lysate with LAMC2 antibody

  • Isolate immune complexes with protein G beads

  • Analyze by Western blot, probing for EGFR

• Deletion Mutation Analysis:

  • Create constructs lacking specific LAMC2 domains (EGF-Lam and LamB regions)

  • Transfect into appropriate cell lines

  • Use LAMC2 antibodies to confirm expression

  • Assess EGFR binding capacity through Co-IP and confocal microscopy

• Subcellular Fractionation:

  • Separate cellular components (membrane, ER, cytosol)

  • Probe fractions with LAMC2 and EGFR antibodies

  • Quantify relative distribution in different conditions

Research has revealed that LAMC2 promotes EGFR membrane deposition by facilitating transport from the ER and preventing protein degradation via ubiquitination. Both the EGF-Lam and LamB regions of LAMC2 are necessary for EGFR receptor binding .

How can FITC-conjugated LAMC2 Antibody be used to evaluate the efficacy of targeted cancer therapies?

LAMC2 expression correlates with response to certain targeted therapies, making it a potential biomarker. To evaluate therapy efficacy:

• Patient-Derived Xenograft (PDX) Models:

  • Establish PDX models from patient tumor samples

  • Administer targeted therapy (e.g., EGFR-TKIs for lung cancer)

  • Harvest tissue at defined timepoints

  • Use LAMC2 Antibody, FITC Conjugated for immunofluorescence analysis

  • Correlate LAMC2 expression patterns with treatment response

• Flow Cytometry Protocol for Monitoring Treatment Response:

  • Harvest cells from treated and untreated samples

  • Fix and permeabilize cells

  • Stain with LAMC2 Antibody, FITC Conjugated

  • Analyze by flow cytometry to quantify changes in expression levels

  • Gate populations based on LAMC2 expression intensity

• Multiplex Imaging:

  • Combine LAMC2 Antibody, FITC Conjugated with antibodies against other markers (e.g., phospho-EGFR, cleaved caspase-3)

  • Use multispectral imaging to assess correlations between LAMC2 and therapy-induced changes

Research indicates that high LAMC2 expression positively correlates with response to gefitinib (EGFR tyrosine kinase inhibitor) treatment in lung cancer, suggesting its potential use as a stratifying biomarker for patients suitable for EGFR-TKI treatment .

What methodologies can address potential cross-reactivity or specificity concerns when using LAMC2 Antibody in multi-parameter experiments?

When designing multi-parameter experiments with LAMC2 Antibody, FITC Conjugated:

• Antibody Validation Protocol:

  • Perform Western blot analysis with the antibody (if unconjugated version available)

  • Verify single band at expected molecular weight (approximately 140 kDa)

  • Test antibody on LAMC2 knockout/knockdown cells as negative control

  • Compare staining pattern with alternative LAMC2 antibody clones

• Spectral Overlap Compensation:

  • When using multiple fluorophores, prepare single-stained controls for each fluorophore

  • Generate compensation matrix to correct for spectral overlap

  • Validate compensation using fluorescence minus one (FMO) controls

• Absorption/Blocking Controls:

  • Pre-incubate LAMC2 Antibody with recombinant LAMC2 protein

  • Apply this mixture in parallel with standard antibody application

  • Compare signal reduction to confirm specificity

• Signal Amplification Alternatives:

  • For weak signals, consider tyramide signal amplification (TSA) rather than increasing antibody concentration

  • This maintains specificity while enhancing detection sensitivity

Careful validation ensures that signals attributed to LAMC2 are genuine, particularly important when investigating complex biological contexts such as ER-mitochondria contacts or cancer signaling pathways.

How can LAMC2 Antibody be used to investigate the role of LAMC2 in the AKT signaling pathway in pancreatic cancer?

Research has shown that LAMC2 regulates a functional FOSL1-AXL axis via AKT phosphorylation in pancreatic ductal adenocarcinoma (PDAC) . To investigate this pathway:

• Phospho-Protein Analysis Protocol:

  • Treat cells with AKT pathway modulators

  • Collect protein lysates at various timepoints

  • Perform Western blot for total and phospho-AKT

  • Use LAMC2 antibodies to correlate LAMC2 expression with AKT activation status

• LAMC2 Knockdown Experimental Design:

  • Generate stable LAMC2 knockdown cell lines using shRNA or CRISPR-Cas9

  • Validate knockdown efficiency using LAMC2 Antibody

  • Assess changes in AKT pathway components (phospho-AKT, FOSL1, AXL)

  • Perform rescue experiments with wild-type LAMC2 transfection

• 3D Organoid Culture Analysis:

  • Establish patient-derived organoids

  • Treat with combination of AXL inhibitors and MEK1/2 inhibitors

  • Perform immunofluorescence with LAMC2 Antibody, FITC Conjugated

  • Quantify growth inhibition and signaling changes

Research demonstrates that LAMC2 inhibition impairs cell cycle progression, induces apoptosis, and sensitizes PDAC to MEK1/2 inhibitors, potentially through modulation of AKT signaling .

What are the technical considerations for optimizing immunofluorescence staining using LAMC2 Antibody, FITC Conjugated in different tissue types?

Optimizing immunofluorescence staining across different tissue types requires systematic consideration of:

• Tissue-Specific Fixation Protocol:

  • Epithelial tissues: 4% paraformaldehyde, 10-15 minutes

  • Connective tissues: Longer fixation (20-30 minutes) may be required

  • Fresh frozen sections: Acetone or methanol fixation, 10 minutes

  • Test multiple fixation conditions to determine optimal protocol for target tissue

• Antigen Retrieval Optimization Matrix:

  Antigen Retrieval Method	Temperature	Duration	Best For
  Citrate buffer (pH 6.0)	95°C	20 min	Most tissues
  EDTA buffer (pH 9.0)	95°C	20 min	Heavily fixed tissues
  Enzymatic (Proteinase K)	37°C	10 min	Tissues with dense ECM

• Blocking and Permeabilization Variables:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Optimize permeabilization (Triton X-100 0.1-0.5%, saponin 0.1%)

  • Extend blocking time for tissues with high background (1-2 hours)

• Antibody Dilution Series:

  • Prepare serial dilutions (1:50, 1:100, 1:200, 1:500)

  • Incubate parallel sections with each dilution

  • Balance optimal signal-to-noise ratio

• Counterstaining Considerations:

  • Select nuclear counterstains with minimal spectral overlap with FITC

  • DAPI or Hoechst 33342 are preferred options

  • Consider additional markers for tissue architecture (e.g., E-cadherin, laminin)

Lung cancer tissues specifically benefit from heat-induced epitope retrieval in citrate buffer (pH 6.0) prior to LAMC2 antibody application, as demonstrated in published protocols .

What are common troubleshooting strategies for weak or absent LAMC2 signal in immunofluorescence experiments?

When encountering weak or absent LAMC2 signal:

• Sequential Troubleshooting Approach:

  • Antibody Functionality Check: Perform dot blot with recombinant LAMC2 protein

  • Fixation Assessment: Test alternative fixatives (PFA, methanol, acetone)

  • Epitope Accessibility: Try different antigen retrieval methods

  • Permeabilization Optimization: Test different detergents and concentrations

  • Signal Amplification: Implement tyramide signal amplification system

• Antibody Concentration Matrix:

  Tissue/Cell Type	Starting Dilution	If Signal Weak	If Background High
  Cell lines	1:200	1:100	1:400
  Fresh tissue	1:100	1:50	1:200
  FFPE tissue	1:50	1:25	1:100

• Incubation Condition Variables:

  • Test both room temperature (1-2 hours) and 4°C overnight incubation

  • Evaluate static vs. gentle agitation during incubation

  • Try humid chamber to prevent section drying

• Photobleaching Prevention:

  • Minimize exposure to light during all steps

  • Use anti-fade mounting medium

  • Image samples promptly after preparation

• Alternative Detection Strategies:

  • If FITC signal remains problematic, consider using unconjugated primary with secondary antibody approach

  • Try signal amplification with biotinylated secondary and streptavidin-fluorophore

Implementation of these systematic approaches helps identify the specific issues affecting LAMC2 detection in experimental systems.

How can researchers validate specificity when using LAMC2 Antibody, FITC Conjugated for studying novel tissue types or disease models?

Validating antibody specificity in novel contexts requires multiple complementary approaches:

• Orthogonal Validation Strategy:

  • Compare with alternative detection methods (Western blot, RNA-seq, qPCR)

  • Use two different antibodies targeting distinct LAMC2 epitopes

  • Correlate expression with known LAMC2-regulated genes

• Genetic Validation Methods:

  • Implement CRISPR/Cas9 knockout of LAMC2

  • Use siRNA knockdown with gradient efficiency

  • Perform rescue experiments with LAMC2 overexpression

  • Employ peptide competition assays

• Tissue-Specific Positive Controls:

  • Identify tissues with known LAMC2 expression patterns

  • Include these as positive controls alongside experimental samples

  • Basement membranes of epithelial tissues typically show high LAMC2 expression

• Cross-Reactivity Assessment:

  • Test antibody on tissues from different species if working with non-human models

  • Check for staining in tissues known to lack LAMC2 expression

  • Perform sequence alignment of immunogen region across species

• Blocking Peptide Confirmation:

  • Pre-incubate antibody with specific blocking peptide

  • Apply to adjacent tissue sections

  • Observe signal reduction or elimination

These validation approaches ensure that observed LAMC2 staining patterns in novel experimental systems accurately reflect protein expression and localization.

What methodological approaches can reveal LAMC2 interactions with binding partners such as MYH9 and MYH10?

To investigate LAMC2 protein interactions with partners like MYH9 and MYH10:

• Co-Immunoprecipitation Protocol Refinements:

  • Use membrane-compatible lysis buffers to preserve native protein interactions

  • Include protease and phosphatase inhibitors

  • Optimize antibody-to-lysate ratios

  • Perform reciprocal IPs (pull down with MYH9/MYH10 antibodies, probe for LAMC2)

• Proximity Ligation Assay (PLA) Workflow:

  • Fix cells under conditions that preserve protein-protein interactions

  • Apply primary antibodies against LAMC2 and binding partner (e.g., MYH9)

  • Add species-specific PLA probes

  • Perform ligation and rolling circle amplification

  • Quantify interaction signals as fluorescent dots

• FRET Analysis Protocol:

  • Transfect cells with fluorescently tagged LAMC2 and binding partners

  • Alternative: Use fluorophore-conjugated antibodies against endogenous proteins

  • Perform acceptor photobleaching FRET

  • Calculate FRET efficiency to determine molecular proximity

• Subcellular Co-localization Quantification:

  • Perform triple immunofluorescence for LAMC2, MYH9/MYH10, and nuclear marker

  • Capture high-resolution confocal z-stacks

  • Calculate Pearson's correlation coefficient and Manders' overlap coefficient

  • Focus analysis on specific subcellular regions (e.g., near nuclear membrane)

Research has demonstrated that LAMC2 co-localizes with both MYH9 and MYH10 near the nuclear membrane, and physical interactions have been confirmed through co-immunoprecipitation experiments .

How should researchers design experiments to study the role of LAMC2 in ER stress and mitochondrial function?

To investigate LAMC2's role in regulating ER stress and mitochondrial function:

• Integrated Experimental Design:

  • Generate LAMC2 knockdown and overexpressing cell lines

  • Apply ER stress inducers (tunicamycin, thapsigargin)

  • Monitor ER stress markers (BiP/GRP78, CHOP, phospho-eIF2α)

  • Assess mitochondrial function (membrane potential, oxygen consumption)

• ER-Mitochondria Contact Site Quantification:

  • Transfect cells with fluorescent organelle markers

  • Apply LAMC2 Antibody, FITC Conjugated for endogenous LAMC2

  • Capture super-resolution images

  • Implement computational analysis of contact site number and length

• Calcium Flux Measurement Protocol:

  • Load cells with calcium indicators (Fluo-4, Rhod-2)

  • Monitor calcium transfer between ER and mitochondria

  • Compare dynamics in LAMC2-manipulated vs. control cells

  • Correlate with ER stress levels

• Mitochondrial Fragmentation Assay:

  • Visualize mitochondrial morphology using MitoTracker

  • Quantify fusion/fission events

  • Assess DRP1 recruitment to mitochondria

  • Determine impact of LAMC2 modulation

Research has shown that LAMC2 co-localizes with both ER and mitochondria at their junction points, and it interacts with DRP1, suggesting a role in regulating ER-mitochondria communication and potentially mitochondrial dynamics .

What controls are essential when using LAMC2 Antibody, FITC Conjugated for quantitative analysis of protein expression?

For rigorous quantitative analysis of LAMC2 expression:

• Essential Controls Table:

  Control Type	Purpose	Implementation
  No primary antibody	Background assessment	Omit LAMC2 antibody, apply all other reagents
  Isotype control	Non-specific binding	Apply FITC-conjugated rabbit IgG at same concentration
  Positive control	Confirm assay function	Include known LAMC2-expressing sample
  Negative control	Validate specificity	Include LAMC2-negative or knockdown sample
  Dynamic range	Validate linearity	Analyze samples with gradient of expression levels

• Standard Curve Generation:

  • Prepare recombinant LAMC2 protein standards at known concentrations

  • Process alongside experimental samples

  • Generate standard curve for absolute quantification

• Normalization Strategy:

  • Include housekeeping protein controls

  • Measure total protein content (BCA assay)

  • Use ratio of LAMC2 to reference protein/total protein

• Technical Replicate Planning:

  • Minimum triplicate technical replicates

  • Randomize sample positioning to avoid position effects

  • Include inter-assay calibrators for multi-batch analyses

• Signal Intensity Verification:

  • Confirm signal falls within linear range of detection system

  • Check for pixel saturation in imaging applications

  • Test serial dilutions to verify antibody response linearity