plac8.1 Antibody, FITC conjugated

What is PLAC8 and what cellular functions does it regulate?

PLAC8 (Placenta-specific gene 8 protein, also known as Protein C15) is a cysteine-rich protein initially identified in placental tissue but now recognized to have diverse functional roles. Research has demonstrated that PLAC8 serves as a critical regulator of multiple cellular processes including:

• Autophagy regulation, particularly in facilitating lysosome-autophagosome fusion

• Cellular proliferation and survival mechanisms, especially in immune cells

• Tumorigenic processes and cancer progression

• Immune cell memory formation, particularly in CD8+ T cells

The protein has a molecular weight corresponding to 110 amino acids and contains unique cysteine-rich domains that contribute to its functional properties . Recent studies have established PLAC8 as a mechanistic link between primary oncogenic mutations (particularly in p53 and RAS) and metabolic reprogramming in pancreatic ductal adenocarcinoma through its regulation of the autophagic machinery .

How does PLAC8 Antibody, FITC conjugated differ from other PLAC8 antibodies?

The PLAC8 Antibody, FITC conjugated represents a specialized reagent that combines targeted recognition of the PLAC8 protein with fluorescent visualization capabilities. Key distinguishing features include:

• Direct conjugation to Fluorescein Isothiocyanate (FITC), eliminating the need for secondary antibody incubation

• Optimized for fluorescence microscopy and flow cytometry applications

• Validated for human PLAC8 detection, specifically targeting recombinant Human Placenta-specific gene 8 protein (amino acids 1-110)

• Polyclonal nature, recognizing multiple epitopes on the PLAC8 protein

Unlike unconjugated PLAC8 antibodies that require additional detection reagents, this FITC-conjugated version allows direct visualization in appropriate fluorescence channels, simplifying experimental workflows and reducing background in multi-color applications.

What is the relationship between PLAC8 and PLAC8L1?

Despite naming similarities, PLAC8 and PLAC8L1 (PLAC8-like 1) represent distinct proteins with different structures and functions:

Antibodies targeting these proteins are not cross-reactive due to significant sequence differences. When conducting research involving either protein, researchers should carefully select antibodies specifically validated for their target of interest .

How does PLAC8 regulate autophagy and what experimental approaches can reveal this mechanism?

PLAC8 functions as a critical mediator of autophagy, particularly at the stage of autophagosome-lysosome (AL) fusion. Experimental evidence demonstrates:

• PLAC8 knockdown results in approximately 80% reduction in GFP-LC3/Lamp2 co-localization in both murine mp53/Ras cells and human CAPAN-2 pancreatic ductal adenocarcinoma cells

• Silencing of PLAC8 leads to accumulation of autophagy markers p62 and LC3 under both normal and nutrient-depleted conditions

• Expression of shRNA-resistant PLAC8 rescues the fusion defect and normalizes p62 and LC3 levels

To investigate PLAC8's role in autophagy, researchers can employ these methodological approaches:

• Fluorescence microscopy with co-localization analysis of GFP-LC3 (autophagosome marker) and LAMP2 (lysosome marker)

• Western blot quantification of p62 and LC3-II accumulation following PLAC8 manipulation

• Autophagic flux assays using Bafilomycin A1 or Chloroquine with and without PLAC8 intervention

• Subcellular fractionation to detect PLAC8 localization to lysosomal compartments

• Live-cell imaging with fluorescently-tagged PLAC8 (using techniques compatible with FITC-conjugated antibodies for endogenous protein)

These approaches can be significantly enhanced using FITC-conjugated PLAC8 antibodies to visualize endogenous PLAC8 localization during autophagic processes.

What role does PLAC8 play in immune cell function and how can PLAC8 antibodies help evaluate this?

PLAC8 demonstrates significant functions in immune cell regulation, particularly in:

• CD8+ T cell memory formation and maintenance

• Monocyte survival, proliferation, and activation during sepsis

• Inflammatory cytokine production regulation

In monocytes, PLAC8 upregulation activates the ERK pathway, leading to:

• Increased phosphorylation of ERK protein

• Elevated expression of CD14 and CD16 (monocyte markers)

• Enhanced production of TNF-α, IL-6, and IL-10

Experimental approaches to study these functions include:

• Flow cytometry with FITC-conjugated PLAC8 antibodies to quantify expression levels in specific immune cell populations

• Cell cycle analysis using PLAC8 antibodies combined with DNA content staining

• Assessment of apoptotic status through Annexin V/PI staining in wild-type versus Plac8-/- cells

• Cytokine profiling following PLAC8 manipulation in immune cells

• Proliferation assays (e.g., CCK-8) to measure cell growth response

The FITC-conjugated PLAC8 antibody is particularly valuable for flow cytometric analyses, allowing simultaneous assessment of PLAC8 expression and other immune markers without additional secondary antibody requirements.

What is the significance of PLAC8 post-translational modifications and how do they affect its function?

Recent research has identified critical post-translational modifications of PLAC8 that significantly impact its stability and function:

• UFMylation (modification by ubiquitin-fold modifier 1) maintains PLAC8 protein stability

• This modification is particularly relevant in triple-negative breast cancer, where PLAC8 is generally highly expressed

• Modified PLAC8 regulates PD-L1 ubiquitination, affecting immune responses in the tumor microenvironment

Experimental approaches to study these modifications include:

• Co-immunoprecipitation assays followed by western blotting to detect UFM1 modification

• Stability assays using cycloheximide chase experiments with wild-type versus mutant PLAC8

• In vitro UFMylation assays to identify specific modification sites

• Immunofluorescence co-localization studies using FITC-conjugated PLAC8 antibodies combined with UFM1 staining

These findings suggest that PLAC8 antibodies capable of distinguishing between modified and unmodified forms could provide valuable insights into the functional consequences of these modifications in different cellular contexts.

What are the optimal protocols for using PLAC8 Antibody, FITC conjugated in flow cytometry?

For optimal flow cytometry results with FITC-conjugated PLAC8 antibody, researchers should consider:

Sample Preparation Protocol:

• Harvest cells (1-5×10^6 cells/sample) and wash twice with ice-cold PBS

• For intracellular staining: Fix cells with 4% paraformaldehyde (10 minutes, room temperature) followed by permeabilization with 0.1% Triton X-100 (5 minutes, room temperature)

• Block with 2% BSA in PBS (30 minutes, 4°C)

• Incubate with FITC-conjugated PLAC8 antibody at optimal dilution (typically 1:50-1:200) for 30-60 minutes at 4°C in the dark

• Wash twice with PBS containing 0.5% BSA

• Resuspend in appropriate flow buffer and analyze promptly

Key Optimization Parameters:

• Titrate antibody to determine optimal concentration (signal-to-noise ratio)

• Include appropriate isotype control (FITC-conjugated Rabbit IgG)

• For apoptosis studies, combine with Annexin V and PI/7-AAD as described in protocols for Plac8-/- experiments

• For cell cycle analysis, add DNA content dye compatible with FITC (such as 7-AAD)

• When studying immune cells, consider using additional markers (CD14, CD16) that correlate with PLAC8 expression

Careful attention to these protocols will ensure specific detection while minimizing background fluorescence.

How should researchers validate specificity of PLAC8 antibody results in experimental systems?

Rigorous validation is essential when using PLAC8 antibodies to ensure experimental results accurately reflect PLAC8 biology:

Recommended Validation Approaches:

• Genetic Controls:

  • Include Plac8 knockout or knockdown samples as negative controls

  • Use cells with verified Plac8 overexpression as positive controls

  • Employ shRNA-resistant PLAC8 rescue systems as described in autophagy studies

• Antibody Validation:

  • Test with recombinant PLAC8 protein in western blot

  • Perform peptide competition assays to confirm specificity

  • Compare results with alternative PLAC8 antibody clones

• Cross-Platform Verification:

  • Confirm PLAC8 expression by at least two independent methods (e.g., flow cytometry and western blot)

  • Correlate protein expression with mRNA levels via qRT-PCR

  • For localization studies, verify with subcellular fractionation

• Controls to Prevent Misinterpretation:

  • Include PLAC8L1 expressing controls to confirm antibody specificity

  • Account for the effect of fixation and permeabilization on epitope accessibility

  • Monitor for potential spectral overlap when using multiple fluorophores

Implementing these validation strategies ensures that experimental findings genuinely reflect PLAC8 biology rather than technical artifacts.

How can PLAC8 antibodies be used to study cancer mechanisms and potential therapeutic targets?

PLAC8 antibodies serve as valuable tools for investigating cancer biology, particularly in:

Cancer Research Applications:

• Triple-Negative Breast Cancer Studies:

  • Assess PLAC8 expression levels, which are generally elevated in triple-negative breast cancer compared to non-triple-negative types

  • Correlate PLAC8 expression with PD-L1 levels to understand immunotherapy response potential

  • Study UFM1 modification patterns of PLAC8 in different breast cancer subtypes

• Pancreatic Ductal Adenocarcinoma Research:

  • Investigate PLAC8's role in autophagy regulation, particularly lysosome-autophagosome fusion

  • Explore connections between RAS/p53 mutations and PLAC8-mediated metabolic reprogramming

  • Assess PLAC8 as a potential therapeutic target or biomarker

• Methodological Approaches:

  • Immunohistochemistry/immunofluorescence of cancer tissues to assess PLAC8 expression patterns

  • Flow cytometry of cancer cell lines to quantify PLAC8 levels and correlate with malignant phenotypes

  • Functional assays following PLAC8 manipulation to measure proliferation, invasion, and drug resistance

The FITC-conjugated PLAC8 antibody is particularly useful for flow cytometric analyses of cancer cell lines and for fluorescence microscopy studies investigating PLAC8 localization in autophagic processes crucial to cancer metabolism.

What protocols are recommended for studying PLAC8 in immune response and memory formation?

For researchers investigating PLAC8's role in immune function, particularly in CD8+ T cell memory formation, the following protocols are recommended:

Immune Function Study Protocols:

• CD8+ T Cell Memory Analysis:

  • Use influenza infection models as described in research by Watford et al.

  • Compare wild-type and Plac8-/- cells using FITC-labeled Annexin V combined with vital dyes (PI or 7-AAD) to distinguish viable, apoptotic, and necrotic cells

  • Monitor effector to memory (ETM) cell transition in the presence or absence of functional PLAC8

• Monocyte Activation Studies:

  • Stimulate PBMCs with LPS and analyze PLAC8 expression by flow cytometry using FITC-conjugated antibodies

  • Correlate PLAC8 levels with ERK phosphorylation and expression of activation markers (CD14, CD16)

  • Measure cytokine production (TNF-α, IL-6, IL-10) in relation to PLAC8 expression levels

• Data Analysis Parameters:

  Parameter	Wild-type Cells	Plac8-deficient Cells	Analysis Method
  Cell survival	Baseline	Decreased	Annexin V/PI staining
  Proliferation	Normal	Reduced	CCK-8 assay, cell cycle analysis
  Cytokine production	TNF-α, IL-6, IL-10	Reduced levels	ELISA
  ERK phosphorylation	Present	Diminished	Western blot
  Memory T cell formation	Efficient	Impaired	Flow cytometry phenotyping

These protocols can be adapted depending on the specific immune cell type and activation conditions being studied.

How can researchers address common technical challenges when using FITC-conjugated PLAC8 antibodies?

When working with FITC-conjugated PLAC8 antibodies, researchers may encounter several technical challenges. Here are solutions to common issues:

Challenge: Weak PLAC8 Signal

• Solution 1: Optimize fixation and permeabilization conditions (test 4% PFA vs. methanol fixation)

• Solution 2: Increase antibody concentration after careful titration experiments

• Solution 3: Extend incubation time (up to overnight at 4°C) while protecting from light

• Solution 4: For intracellular epitopes, ensure adequate permeabilization with appropriate detergents

Challenge: High Background/Non-specific Staining

• Solution 1: Increase blocking time and concentration (try 5-10% normal serum)

• Solution 2: Include FcR blocking reagent when working with immune cells

• Solution 3: Reduce antibody concentration after proper titration

• Solution 4: Filter all buffers to remove particulates that may bind antibody non-specifically

Challenge: FITC Photobleaching

• Solution 1: Minimize exposure to light during all procedures

• Solution 2: Use anti-fade mounting media for microscopy applications

• Solution 3: Analyze flow cytometry samples promptly after staining

• Solution 4: Consider using higher-stability fluorophores if persistent issues occur

Challenge: Autofluorescence Interference

• Solution 1: Include unstained controls to establish baseline autofluorescence

• Solution 2: Use spectral compensation when analyzing multiple fluorophores

• Solution 3: Consider alternative fluorophores if tissue/cell autofluorescence overlaps significantly with FITC

Addressing these technical challenges will help ensure reliable and reproducible results when using FITC-conjugated PLAC8 antibodies.

How should researchers interpret discrepancies between PLAC8 antibody data and functional outcomes?

When faced with discrepancies between PLAC8 antibody staining results and functional outcomes, researchers should consider these analytical approaches:

Discrepancy Analysis Framework:

• Epitope Accessibility Issues:

  • Different antibodies may recognize distinct epitopes that could be masked by protein interactions or conformational changes

  • Compare results with antibodies targeting different PLAC8 epitopes

  • Consider whether post-translational modifications (especially UFM1) might affect antibody binding

• Functional Threshold Effects:

  • Establish dose-response relationships between PLAC8 expression levels and functional outcomes

  • Determine whether a minimum threshold of PLAC8 is required for specific functions

  • Consider that partial knockdown may be insufficient to observe phenotypic changes seen in complete knockouts

• Context-Dependent Activity:

  • PLAC8 functions differently in various cellular contexts (e.g., cancer cells vs. immune cells)

  • Cell-specific interaction partners may modulate PLAC8 activity

  • Verify the presence of relevant PLAC8 interaction partners in your experimental system

• Technical Verification Steps:

  • Confirm antibody specificity using multiple controls (as outlined in section 3.2)

  • Verify protein expression using alternative methods (western blot, PCR)

  • Use functional rescue experiments with shRNA-resistant PLAC8 constructs to confirm specificity of observed phenotypes

Careful analysis using this framework can help resolve apparent discrepancies and lead to more accurate interpretation of experimental results involving PLAC8.

What experimental design considerations are critical when investigating PLAC8's multiple cellular functions?

Given PLAC8's involvement in diverse cellular processes, thoughtful experimental design is crucial:

Critical Experimental Design Elements:

• Cell Type Selection:

  • Choose appropriate cellular models based on research question (cancer cells for autophagy studies , immune cells for memory studies )

  • Consider using primary cells where possible for physiological relevance

  • Include cell lines with known PLAC8 expression levels as reference points

• Temporal Considerations:

  • Design time-course experiments to capture dynamic PLAC8-dependent processes

  • For autophagy studies, include both basal and starvation-induced conditions

  • For immune response studies, examine both acute activation and memory phases

• Mechanistic Dissection Approach:

  • Use specific inhibitors to delineate PLAC8-dependent pathways:

    • Autophagy inhibitors (Bafilomycin A1, Chloroquine)

    • ERK pathway inhibitors (U0126, PD98059)

    • UFMylation pathway inhibitors

  • Employ genetic approaches (CRISPR/Cas9, siRNA, shRNA) with appropriate controls

• Integrated Data Analysis:

  • Correlate PLAC8 expression with multiple functional readouts simultaneously

  • Consider systems biology approaches to map PLAC8 interaction networks

  • Use mathematical modeling to integrate disparate data types

• Validation Across Systems:

  • Verify findings in multiple cell lines/types

  • Translate in vitro findings to relevant in vivo models

  • When possible, confirm results in human samples to establish clinical relevance