plac8.2 Antibody, FITC conjugated

What is plac8.2 and what is its relationship to human PLAC8?

plac8.2 (also known as Zgc:158845 protein) is a zebrafish (Danio rerio) protein that belongs to the PLAC8 family. Human PLAC8 (placenta-specific gene 8 protein, also known as Protein C15) is a 12 kDa protein initially identified in placental tissue. The plac8.2 protein represents one of the zebrafish orthologs of the human PLAC8. Research has implicated PLAC8 family proteins in diverse biological processes including cell proliferation, autophagy, and immune responses .

What is known about the structure and specifications of commercially available plac8.2 Antibody, FITC conjugated?

The plac8.2 Antibody, FITC conjugated is a rabbit polyclonal antibody specifically raised against a peptide sequence from Zebrafish Zgc:158845 protein (amino acids 5-19) . Its reactivity is primarily with zebrafish samples. The antibody is provided in liquid form, with a buffer composition of PBS containing 0.03% Proclin 300 as a preservative and 50% glycerol at pH 7.4. The product undergoes Protein G purification with purity greater than 95%. For long-term storage, the manufacturer recommends storing at -20°C or -80°C and avoiding repeated freeze-thaw cycles .

How should I design experiments to investigate plac8.2 expression patterns in zebrafish embryonic development?

When investigating plac8.2 expression patterns during zebrafish development, a comprehensive experimental design should include:

For quantitative analysis, consider dissociating embryos at different stages for flow cytometry to quantify the percentage of plac8.2-positive cells and their fluorescence intensity .

What controls should be included when using plac8.2 Antibody, FITC conjugated in immunofluorescence studies?

A rigorous immunofluorescence experiment using plac8.2 Antibody, FITC conjugated requires the following controls:

These controls help distinguish between specific signal and artifacts, which is crucial for accurate interpretation of plac8.2 localization and expression patterns .

How do I optimize the dilution of plac8.2 Antibody, FITC conjugated for flow cytometry applications?

To determine the optimal antibody dilution for flow cytometry:

• Prepare single-cell suspensions from zebrafish tissues known to express plac8.2

• Aliquot equal numbers of cells (typically 1×10^6 cells per tube) into multiple samples

• Test a titration series of antibody dilutions:

• Include controls listed in question 2.2

• Incubate cells with antibody for 30-60 minutes on ice in the dark

• Wash cells twice with flow cytometry buffer (PBS with 1-2% FBS)

• Analyze samples, recording:

  • Mean/median fluorescence intensity

  • Signal-to-noise ratio (comparing to isotype control)

  • Percentage of positive cells

• Plot a titration curve showing signal-to-noise ratio versus antibody concentration

• Select the dilution at the beginning of the plateau phase of the curve

The optimal dilution provides maximum specific signal with minimal background and economical antibody usage .

How can I use plac8.2 Antibody, FITC conjugated to investigate potential functional parallels between zebrafish plac8.2 and human PLAC8 in immune responses?

Based on human PLAC8's role in immune function and sepsis , you can design comparative studies to investigate functional conservation:

• Baseline expression profiling:

  • Use flow cytometry with plac8.2 Antibody, FITC conjugated to characterize expression in zebrafish immune cell populations

  • Compare distribution patterns with known human PLAC8 expression in immune cells

  • Perform dual immunofluorescence with markers for macrophages, neutrophils, and lymphocytes

• Immune challenge experiments:

  • Design parallel experiments in zebrafish and human cell models:

• ERK pathway analysis:

  • Investigate if zebrafish plac8.2, like human PLAC8, activates the ERK pathway in immune cells

  • Test ERK inhibitors (e.g., GDC-0994) and activators (e.g., EGF) in both systems

  • Monitor phosphorylation of ERK1/2 following plac8.2/PLAC8 manipulation

• Genetic manipulation approaches:

  • Generate plac8.2 knockout zebrafish using CRISPR/Cas9

  • Compare immune phenotypes to human PLAC8-deficient cells

  • Look for conserved functions in phagocytosis, cytokine production, and cell survival

This cross-species approach will reveal whether the plac8.2-ERK pathway in zebrafish functionally parallels the PLAC8-ERK interaction observed in human sepsis models .

What role does plac8.2 play in autophagy, and how can this be studied using the FITC-conjugated antibody?

Based on the known role of human PLAC8 in autophagy , you can design experiments to investigate plac8.2's role in zebrafish autophagy:

• Colocalization with autophagy markers:

  • Perform dual immunofluorescence with plac8.2 Antibody, FITC conjugated and antibodies against LC3-B (autophagosome marker)

  • Analyze colocalization under basal conditions and after autophagy induction

  • Quantify Pearson's correlation coefficient between plac8.2 and LC3-B signals

• Autophagy induction experiments:

• Genetic approaches:

  • Knockdown plac8.2 using morpholinos or CRISPR/Cas9

  • Monitor changes in autophagy markers (LC3-II/LC3-I ratio)

  • Assess autophagic flux using tandem-fluorescent LC3 reporters

  • Rescue experiments with human PLAC8 to test functional conservation

• Cadmium exposure model:

  • Expose zebrafish to cadmium (similar to studies in human cells)

  • Monitor plac8.2 expression using the FITC-conjugated antibody

  • Assess autophagy induction and correlation with plac8.2 levels

  • Test if plac8.2 knockdown affects cadmium-induced autophagy

Understanding plac8.2's role in autophagy could reveal conserved mechanisms between zebrafish and humans, potentially informing studies on cellular stress responses and heavy metal toxicity .

How can I correlate plac8.2 protein expression with gene expression data in zebrafish models?

To establish meaningful correlations between protein and mRNA expression:

• Parallel sample analysis:

  • Divide samples for protein detection with plac8.2 Antibody, FITC conjugated and RNA extraction for qRT-PCR

  • Process samples from the same experimental conditions and timepoints

  • Generate paired data points for correlation analysis

• Sequential analysis in tissue sections:

  • Document plac8.2 protein localization using immunofluorescence

  • Perform in situ hybridization on the same or adjacent sections

  • Use computational image alignment to correlate protein and mRNA signals

• Time-course experiments:

• Cell-type specific analysis:

  • Sort cell populations based on specific markers

  • Analyze plac8.2 protein and mRNA in each population

  • Identify cell types with concordant or discordant expression patterns

• Validation with genetic manipulation:

  • Use siRNA to knockdown plac8.2 (as done in human cell models)

  • Confirm knockdown efficiency at both mRNA (qRT-PCR) and protein levels (antibody staining)

  • Calculate the relationship between knockdown efficiency at mRNA and protein levels

These approaches will reveal the dynamics of plac8.2 expression regulation and potential post-transcriptional mechanisms affecting protein abundance relative to mRNA levels .

What are the common causes of high background when using plac8.2 Antibody, FITC conjugated, and how can they be addressed?

High background is a common challenge with fluorescent antibodies. Here are systematic solutions:

For zebrafish embryos specifically, the yolk is highly autofluorescent in the green spectrum. Consider deyolking embryos when possible or using longer wavelength fluorophores (if available in alternative antibody formats) .

How can I validate the specificity of plac8.2 Antibody, FITC conjugated in zebrafish models?

Comprehensive validation of antibody specificity requires multiple approaches:

• Genetic validation:

  • Use plac8.2 knockout or knockdown zebrafish

  • Signal should be significantly reduced or eliminated

  • Method: CRISPR/Cas9 gene editing or morpholino knockdown

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (5-19AA sequence)

  • Run parallel staining with blocked and unblocked antibody

  • Specific signal should disappear in the blocked sample

• Western blot validation:

  • Confirm detection of a single band at expected molecular weight (similar to human PLAC8 at 12-13 kDa)

  • Test multiple tissues with varying expression levels

  • Compare to mRNA expression pattern from public databases

• Cross-validation with different detection methods:

• Heterologous expression:

  • Express tagged plac8.2 in a non-zebrafish system

  • Confirm antibody detection of the recombinant protein

  • Compare with endogenous protein detection

These validation approaches ensure that observed signals truly represent plac8.2 protein rather than artifacts or cross-reactivity .

What are the best practices for storing and handling plac8.2 Antibody, FITC conjugated to maintain its activity?

FITC-conjugated antibodies require special handling to maintain fluorescence intensity and binding capacity:

FITC is particularly sensitive to high pH environments, so maintain buffer pH around 7.2-7.4. To assess potential activity loss, periodically test the antibody on a positive control sample and monitor signal intensity over time .

How should I analyze plac8.2 expression patterns across different zebrafish tissues quantitatively?

Comprehensive quantitative analysis requires systematic approaches:

• Flow cytometry analysis:

  • Prepare single-cell suspensions from different tissues

  • Stain with plac8.2 Antibody, FITC conjugated using standardized protocol

  • Analyze and quantify:

• Immunofluorescence quantification:

  • Image multiple fields from each tissue section

  • Use image analysis software (ImageJ/Fiji) to quantify:

  • Total FITC integrated density

  • Area of positive staining

  • Mean pixel intensity in positive regions

  • Normalize to cell number (DAPI+ nuclei)

• Statistical analysis:

  • Perform multiple biological replicates (n≥3)

  • Apply appropriate tests:

    • ANOVA with post-hoc tests for multi-tissue comparison

    • t-tests for pairwise comparisons

  • Present with error bars (standard deviation or standard error)

  • Report p-values and significance levels

• Visualization methods:

  • Heatmaps showing expression across tissues

  • Box plots showing distribution and outliers

  • Bar graphs with error bars for comparing means

These quantitative approaches enable objective comparison of plac8.2 expression patterns and correlation with functional data .

How do I interpret apparent contradictions between plac8.2 protein levels detected by the antibody and mRNA expression data?

Discrepancies between protein and mRNA levels are common and biologically meaningful:

• Biological explanations:

• Technical considerations:

  • Antibody specificity issues (verify with controls from question 4.2)

  • RNA probe cross-reactivity with related transcripts

  • Different detection sensitivities between methods

  • Sample preparation differences affecting protein vs. mRNA preservation

• Validation approaches:

  • Perform pulse-chase experiments to determine protein half-life

  • Use translation inhibitors to correlate mRNA and protein dynamics

  • Examine subcellular localization of protein vs. mRNA

  • Use alternative detection methods to confirm observations

• Functional relevance:

  • Determine if biological activity correlates better with protein or mRNA levels

  • Assess if post-transcriptional regulation might be physiologically significant

  • Consider if the discrepancy reveals regulatory mechanisms specific to plac8.2

Understanding these discrepancies can provide insights into the regulation of plac8.2 and potentially identify novel post-transcriptional mechanisms .

How can I use plac8.2 antibody data to understand potential functional conservation between zebrafish and human PLAC8 proteins?

To evaluate functional conservation between species:

• Expression pattern comparison:

  • Map plac8.2 expression in zebrafish tissues using FITC-conjugated antibody

  • Compare with human PLAC8 expression patterns from literature

  • Identify anatomically equivalent tissues showing similar expression

• Functional domain analysis:

  • Determine if antibody epitope corresponds to conserved functional domains

  • Correlate staining patterns with protein structure predictions

  • Consider if posttranslational modifications affect epitope recognition

• Response to stimuli:

• Genetic rescue experiments:

  • Knockdown zebrafish plac8.2

  • Attempt rescue with human PLAC8 expression

  • Quantify restoration of phenotype and molecular markers

  • This provides functional evidence of conservation

• Pathway interaction conservation:

  • Investigate if plac8.2 interacts with the ERK pathway in zebrafish, similar to human PLAC8

  • Examine if plac8.2 regulates autophagy in response to stress, similar to human PLAC8

  • Test if plac8.2 affects the same downstream targets (cytokines, proliferation markers)

These approaches can reveal functional conservation despite species differences and potential divergence in specific regulatory mechanisms .