ARPC5B Antibody

What is ARPC5 and what role does it play in cellular function?

ARPC5 is a subunit of the Arp2/3 complex that drives the formation of branched actin networks essential for many cellular processes. In humans, the ARPC5 subunit is encoded by two paralogous genes (ARPC5 and ARPC5L) that share 67% identity . The Arp2/3 complex has a unique ability to nucleate actin filaments at an angle from preexisting filaments, resulting in a branched network of polymerized actin that is crucial for cellular functions including migration, phagocytosis, and immune synapse formation . ARPC5 deficiency has been shown to affect actin cytoskeleton organization and function in vitro, demonstrating its essential role in cellular architecture and motility .

How should researchers validate ARPC5 antibodies for experimental applications?

For proper ARPC5 antibody validation, researchers should:

• Confirm antibody specificity through Western blot analysis comparing wild-type cells with ARPC5-deficient cells (either genetic knockout models or siRNA-treated cells)

• Verify antibody performance in multiple applications (immunoblotting, immunoprecipitation, immunofluorescence) with appropriate positive and negative controls

• Test cross-reactivity with the paralog ARPC5L to ensure specificity between the two closely related proteins

• Use multiple antibodies targeting different epitopes of ARPC5 to confirm findings

• Include loading controls like GAPDH (1:10000 dilution) when performing Western blots, with ARPC5 antibodies typically used at 1:600 dilution

What are the optimal sample preparation techniques for ARPC5 detection in Western blotting?

For optimal Western blot detection of ARPC5:

• Extract total protein using RIPA buffer containing protease inhibitors

• Quantify protein concentration using a BCA protein assay kit

• Separate approximately 20 μg of protein using 12% SDS-PAGE

• Transfer proteins to PVDF membranes and block with 5% nonfat milk for 1 hour

• Incubate with ARPC5 primary antibody (1:600 dilution) overnight at 4°C

• Incubate with appropriate secondary antibody (1:2000 dilution) for 1 hour

• Visualize using a chemiluminescent imaging system

This protocol ensures optimal detection of ARPC5 while minimizing background noise and non-specific binding.

How can researchers distinguish between ARPC5 and ARPC5L in experimental analyses?

Distinguishing between ARPC5 and ARPC5L requires careful experimental design:

• Use paralog-specific antibodies that target non-conserved regions of these proteins

• Validate antibody specificity using overexpression and knockdown studies for each paralog

• Implement parallel immunoblotting with antibodies specific to each paralog to compare expression levels

• Consider complementary RNA-based approaches (RT-qPCR with paralog-specific primers) to correlate protein detection with mRNA expression

• When analyzing protein complexes, use immunoprecipitation followed by mass spectrometry to identify peptides unique to each paralog

Research indicates that these paralogs have distinct biological roles even within the same complex, with ARPC5L showing a partially disordered N-terminal region compared to ARPC5, which may contribute to functional differences in Arp2/3 complex activity .

What experimental approaches are recommended for studying ARPC5 deficiency models?

When investigating ARPC5 deficiency models, researchers should consider:

• CRISPR/Cas9-mediated approaches to create cell lines with ARPC5 knockout

• Parallel analysis of ARPC5L expression to assess compensatory mechanisms

• Functional assays for actin cytoskeleton organization including phalloidin staining and live-cell imaging

• Migration and invasion assays to evaluate cellular motility defects

• Embryonic development analysis in animal models, as homozygous Arpc5−/− mice do not survive past embryonic day 9 due to developmental defects

• Immunoblotting for other Arp2/3 complex components to evaluate complex stability

• Rescue experiments with wild-type ARPC5 to confirm phenotype specificity

Studies have demonstrated that ARPC5 loss affects actin cytoskeleton organization despite upregulation of its paralog ARPC5L, indicating non-redundant functions .

How can ARPC5 antibodies be utilized in immune cell research?

For immune cell investigations with ARPC5 antibodies:

• Analyze ARPC5 expression across different immune cell subsets using flow cytometry with cell-specific markers

• Implement immunofluorescence microscopy to visualize ARPC5 localization during immune synapse formation

• Use time-lapse imaging with fluorescently tagged ARPC5 antibodies to track dynamics during immune cell activation

• Compare ARPC5 versus ARPC5L expression patterns in different immune cell populations

• Investigate ARPC5's role in phagocytosis and migration through functional knockdown studies combined with antibody-based detection methods

Research has shown that ARPC5 deficiency is associated with severe immunological defects, including recurrent infections, early-onset autoimmunity, and inflammation, emphasizing its critical role in immune function .

What are the key considerations when using ARPC5 antibodies for cancer research applications?

For oncology research applications of ARPC5 antibodies:

• Employ tissue microarrays with ARPC5 antibodies to evaluate expression across tumor types and correlate with clinicopathological features

• Combine with patient survival data to assess prognostic value, as high ARPC5 expression correlates with poor prognosis in glioma patients

• Use multiplexed immunofluorescence to simultaneously detect ARPC5 and immune cell markers like CD3 to evaluate correlations with tumor-infiltrating lymphocytes

• Implement ARPC5 knockdown studies in cancer cell lines followed by proliferation and invasion assays

• Analyze the relationship between ARPC5 expression and response to immunotherapy based on tumor mutation burden (TMB) and microsatellite instability (MSI) status

Research indicates that ARPC5 expression is significantly overexpressed in gliomas and strongly associated with immune scores, infiltrating immune cells, and immunotherapy outcomes .

What methodology should be employed to investigate ARPC5's role in the Arp2/3 complex assembly?

To study ARPC5's role in Arp2/3 complex assembly:

• Perform co-immunoprecipitation studies using ARPC5 antibodies to pull down associated complex components

• Analyze complex formation through size-exclusion chromatography followed by immunoblotting with ARPC5 antibodies

• Use sucrose gradient ultracentrifugation to separate intact complexes from free subunits

• Implement structural studies including cryo-EM with antibody-based labeling to locate ARPC5 within the complex

• Conduct comparative analyses between complexes containing ARPC5 versus ARPC5L

Research has shown that ARPC5 deficiency leads to reduced expression of interacting proteins ARPC1B and ARPC1A while not affecting other Arp2/3 complex components, suggesting a hierarchical assembly process dependent on ARPC5 .

How can researchers interpret contradictory findings between ARPC5 mRNA and protein expression?

To reconcile discrepancies between ARPC5 mRNA and protein expression:

• Parallel analysis of mRNA (RT-qPCR) and protein levels (Western blot) across multiple cell types and tissues

• Evaluation of post-transcriptional regulation mechanisms including microRNA targeting and RNA binding protein effects

• Assessment of protein stability through cycloheximide chase experiments

• Analysis of alternative splice variants that may be detected at mRNA level but not recognized by antibodies

• Consideration of technical limitations, including antibody sensitivity and primer efficiency

Research has shown cases where ARPC5 mRNA was detected, but expression of ARPC5 protein was not observed in patient-derived cells, highlighting the importance of comprehensive analysis at both RNA and protein levels .

What are common challenges when using ARPC5 antibodies and how can they be addressed?

Common challenges with ARPC5 antibodies and their solutions include:

• Cross-reactivity with ARPC5L:

  • Use antibodies targeting non-conserved regions

  • Validate with knockout/knockdown controls

  • Employ peptide competition assays to confirm specificity

• Weak signal detection:

  • Optimize antibody concentration (starting with 1:600 dilution)

  • Extend primary antibody incubation time to overnight at 4°C

  • Use signal enhancement systems compatible with your detection method

• Inconsistent results between applications:

  • Validate each antibody for specific applications (WB, IF, IHC, IP)

  • Optimize fixation methods (particularly important for preserving actin cytoskeleton structures)

  • Adjust blocking conditions to minimize background while preserving epitope recognition

• Difficulty detecting complexed ARPC5:

  • Use non-denaturing conditions for native complex preservation

  • Consider epitope accessibility in assembled Arp2/3 complex

  • Implement proximity ligation assays to detect ARPC5 within intact complexes

What controls should be included when evaluating ARPC5 expression in disease models?

Essential controls for ARPC5 expression studies in disease models:

• Positive controls:

  • Cell lines with verified high ARPC5 expression

  • Recombinant ARPC5 protein for antibody validation

  • Tissues known to express ARPC5 (based on Human Protein Atlas data)

• Negative controls:

  • CRISPR/Cas9-generated ARPC5 knockout cells

  • siRNA-mediated ARPC5 knockdown samples

  • Isotype control antibodies to assess non-specific binding

• Experimental controls:

  • Matched healthy vs. diseased tissues processed identically

  • Expression analysis of other Arp2/3 complex components

  • Parallel assessment of ARPC5L to evaluate compensatory expression

  • GAPDH or other housekeeping controls for normalization

Research shows that ARPC5 expression needs careful control validation, especially when evaluating its potential as a biomarker in conditions like glioma .

How can single-cell analysis techniques be combined with ARPC5 antibodies for heterogeneity studies?

For integrating ARPC5 antibodies with single-cell analysis:

• Implement CyTOF (mass cytometry) with metal-conjugated ARPC5 antibodies for high-dimensional analysis of ARPC5 expression across cell populations

• Use single-cell Western blotting techniques to quantify ARPC5 levels in individual cells

• Apply CODEX multiplexed imaging to simultaneously visualize ARPC5 and multiple cell markers in tissue sections

• Combine single-cell RNA-seq with protein validation using ARPC5 antibodies on sorted cell populations

• Implement spatial transcriptomics approaches complemented by ARPC5 immunostaining on adjacent tissue sections

Single-cell analysis has revealed that ARPC5 expression is increased in specific cell types including astrocytes, monocytes, and T cells in the context of glioma .

What are the optimal approaches for studying ARPC5 dynamics in live cells?

For investigating ARPC5 dynamics in living cells:

• Generate cell lines expressing fluorescently tagged ARPC5 (ensuring tag location doesn't interfere with complex assembly)

• Use antibody fragments (Fab) conjugated with fluorophores for live-cell imaging of endogenous ARPC5

• Implement FRAP (Fluorescence Recovery After Photobleaching) to measure ARPC5 turnover rates within the Arp2/3 complex

• Apply lattice light-sheet microscopy for high-resolution 3D imaging of ARPC5 dynamics during cell migration

• Use optogenetic approaches combined with ARPC5 visualization to manipulate and monitor actin dynamics simultaneously

These techniques allow researchers to visualize the spatial and temporal dynamics of ARPC5 during processes like immune synapse formation, phagocytosis, and cell migration.

How can ARPC5 antibodies contribute to understanding the pathophysiology of ARPC5 deficiency disorders?

ARPC5 antibodies can advance understanding of ARPC5 deficiency disorders through:

• Immunohistochemical analyses of developmental tissues to identify affected structures and pathways

• Flow cytometric evaluation of immune cell subsets with ARPC5 antibodies to identify specific lineages affected by ARPC5 deficiency

• Investigation of cytokine signaling pathways, particularly IL-6 signaling which is distinctively impacted in ARPC5 deficiency

• Combined analysis with markers of actin cytoskeleton to characterize the "actinopathy" phenotype

• Screening of patient samples to identify potential cases of partial ARPC5 deficiency that might present with milder phenotypes

Research has shown that ARPC5 deficiency leads to a syndrome featuring immune disease, multiple congenital anomalies, and early postnatal death, highlighting the necessity for comprehensive antibody-based detection methods .

What emerging technologies might enhance ARPC5 antibody applications in research?

Emerging technologies with potential to advance ARPC5 antibody applications:

• Super-resolution microscopy techniques such as STORM and PALM for nanoscale visualization of ARPC5 within the Arp2/3 complex

• Proximity-dependent biotinylation (BioID) combined with ARPC5 antibodies to identify transient interaction partners

• Cryo-electron tomography with antibody-based gold labeling for in situ structural studies of the Arp2/3 complex

• Organ-on-chip models combined with ARPC5 immunodetection to study dynamic cellular processes under physiologically relevant conditions

• CRISPR activation/interference systems to modulate ARPC5 levels while monitoring effects with validated antibodies

These technologies could provide unprecedented insights into the spatial organization, dynamic interactions, and functional consequences of ARPC5 within various cellular contexts.

How might ARPC5 antibodies contribute to the development of diagnostic tools for ARPC5-related disorders?

Potential diagnostic applications of ARPC5 antibodies include:

• Development of immunoassays for screening patients with suspected actinopathies or immunodeficiencies

• Creation of tissue-based diagnostic panels combining ARPC5 with other actin cytoskeleton markers

• Implementation in flow cytometry-based diagnostics for rapid assessment of ARPC5 expression in immune cells

• Application in prenatal testing for families with known ARPC5 mutations

• Utilization in prognostic assessments for cancers where ARPC5 expression correlates with outcome