WDR83 Antibody

What is WDR83 and why is it an important research target?

WDR83 (WD repeat domain-containing protein 83), also known as MORG1 (mitogen-activated protein kinase organizer 1), is a member of the WD-40 protein family . This protein functions as a molecular scaffold for various multimeric protein complexes and associates with several components of the extracellular signal-regulated kinase (ERK) pathway, promoting ERK activity in response to serum or other signals . Additionally, WDR83 interacts with egl nine homolog 3 (EGLN3, also known as PHD3) and regulates expression of hypoxia-inducible factor 1 . Research has shown that WDR83 is involved in bidirectional regulation with deoxyhypusine synthase (DHPS), forming an RNA duplex at overlapping 3′ untranslated regions that increases their mutual stability . This pair of protein-coding cis-sense/antisense transcripts has been found to be upregulated in gastric cancer and other cancers, promoting cell proliferation .

The design of immunogens for WDR83 antibodies follows careful selection criteria to ensure specificity. According to the Human Protein Atlas project, "The region with the lowest possible identity is always selected for antigen design, with a maximum identity of 60% allowed for designing a single-target antigen" .

Different manufacturers use various immunogen strategies:

• Fusion proteins corresponding to regions derived from internal residues of human WD repeat domain 83

• Synthetic peptides derived from the internal region of human WDR83 (amino acids 141-190)

• Peptide sequences such as "CLSERDTHVVSCSEDGKVFFWDLVEGALALALPVGSGVVQSLAYHPTEPCLLTAMGGSVQCWREEAYEAEDGAG" for specific epitope targeting

These careful immunogen designs help minimize cross-reactivity with other WD-repeat containing proteins, which is crucial for experimental specificity .

What are the optimal conditions for using WDR83 antibodies in immunohistochemistry (IHC)?

For optimal immunohistochemistry results with WDR83 antibodies, researchers should consider the following protocol parameters based on validated applications:

• Antibody dilution: Most effective dilutions range from 1:30 to 1:1000 depending on the specific antibody and tissue type

• Sample preparation: Paraffin-embedded tissues have been successfully used for WDR83 IHC staining

• Antigen retrieval: Though not explicitly stated in all sources, standard heat-induced epitope retrieval is likely necessary

• Detection system: Secondary antibodies conjugated to HRP, AP, or fluorophores are compatible with primary WDR83 antibodies

• Validated tissue types: Human thyroid cancer tissue and human colon cancer tissue have shown successful staining

The Prestige Antibodies from Sigma-Aldrich have been extensively validated on a tissue array of 44 normal human tissues and 20 of the most common cancer types, providing broader validation data for various tissue applications .

What protocols yield optimal results when using WDR83 antibodies for Western blotting?

For Western blotting applications with WDR83 antibodies, researchers should follow these guidelines for optimal results:

• Antibody concentration: Recommended concentrations range from 0.04-0.4 μg/mL for Prestige antibodies to dilutions of 1:500-1:1000 for other commercial antibodies

• Expected molecular weight: WDR83 typically appears around 34 kDa on Western blots, though the observed molecular weight may differ from the calculated weight

• Sample types: Validated sample types include tissue homogenates and cell lysates from human, mouse, and rat origins

• Blocking conditions: While specific blocking conditions vary between manufacturers, standard 5% non-fat milk or BSA in TBST is likely appropriate

• Detection systems: HRP-conjugated secondary antibodies (anti-rabbit IgG) are commonly used

It's worth noting that for antibodies with uncertain results, revalidation using an over-expression lysate is sometimes performed to confirm specificity , suggesting this as a valuable control strategy.

How should WDR83 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of WDR83 antibodies is crucial for maintaining their activity and specificity:

WDR83 antibodies are typically shipped either with ice packs at 4°C or wet ice , and should be stored immediately upon receipt. Most formulations contain stabilizers such as glycerol (50%), sodium azide (0.05%), or other preservatives to maintain antibody integrity .

When working with lyophilized antibodies, reconstitution should follow manufacturer guidelines, typically with distilled water or buffer to yield concentrations between 0.5-1.3 mg/mL .

How can WDR83 antibodies be used to investigate the bidirectional regulation between WDR83 and DHPS?

Investigation of the bidirectional regulation between WDR83 and DHPS requires sophisticated experimental approaches utilizing WDR83 antibodies:

• RNA-protein complex immunoprecipitation: WDR83 antibodies can be used to pull down RNA-protein complexes, followed by RT-PCR to detect DHPS mRNA, establishing their physical association in cellular contexts .

• Dual silencing experiments: As demonstrated in prior research, "To study whether there was a possible regulation of DHPS by WDR83, DHPS mRNA and protein expression were examined by silencing WDR83" . This approach requires WDR83 antibodies for Western blotting to confirm knockdown efficiency and to monitor effects on DHPS protein levels.

• Co-localization studies: Immunofluorescence with WDR83 antibodies (dilution 0.25-2 μg/mL ) combined with DHPS detection can reveal subcellular sites of interaction.

• Overexpression validation: Expression constructs for WDR83 followed by Western blotting can confirm the reciprocal regulation pattern with DHPS, providing a cellular model for studying this regulatory mechanism .

This bidirectional regulation has significant implications for cancer research, as this pair of genes shows positive correlation in gastric cancer and other cancers, driving pathophysiology by promoting cell proliferation .

What approaches can be used to validate WDR83 antibody specificity for advanced research applications?

Rigorous validation of WDR83 antibody specificity is essential for reliable research results. The Human Protein Atlas and commercial suppliers recommend multiple validation strategies:

• Enhanced validation methodologies:

  • Genetic validation: siRNA knockdown followed by antibody staining to confirm signal reduction

  • Recombinant expression validation: Testing antibodies on cell lines with and without recombinant expression of the target protein

  • Independent antibody validation: Comparing staining patterns of two or more antibodies directed against different epitopes of WDR83

  • Orthogonal validation: Correlation between protein and mRNA expression levels

  • Capture MS validation: Mass spectrometry confirmation of immunoprecipitated proteins

• Protein array testing: Some WDR83 antibodies are validated on protein arrays containing 384 different antigens including the antibody target to analyze cross-reactivity .

• Western blot validation: Testing across multiple human tissues and cell lines to evaluate antibody specificity, with particular attention to the expected 34 kDa band .

• Tissue microarray validation: Comprehensive testing across 44 normal human tissues and 20 common cancer types provides robust validation across diverse biological contexts .

For the most stringent applications, researchers should consider performing multiple validation methods before proceeding with critical experiments.

How can WDR83 antibodies be utilized to study its role in MAPK signaling pathways?

WDR83 (MORG1) functions as a mitogen-activated protein kinase organizer, making its study crucial for understanding MAPK signaling pathways. Researchers can employ WDR83 antibodies in several sophisticated approaches:

• Co-immunoprecipitation studies: Using WDR83 antibodies to pull down protein complexes, followed by Western blotting for ERK pathway components can reveal direct protein-protein interactions and scaffold functions .

• Phosphorylation state analysis: Combining WDR83 immunoprecipitation with phospho-specific antibodies against ERK pathway components can elucidate how WDR83 modulates signaling activation states.

• Stimulus-response experiments: Treating cells with serum or other MAPK-activating signals, followed by temporal analysis of WDR83-associated proteins using immunoprecipitation and Western blotting can reveal dynamic signaling complex formation .

• Subcellular localization studies: Immunofluorescence using WDR83 antibodies (1:500-1:1000 dilution for IHC ) combined with markers for subcellular compartments can reveal translocation events during signaling activation.

• Scaffold disruption experiments: Introducing competing peptides based on the WDR83 immunogen sequences, followed by analysis of MAPK pathway activation can demonstrate the functional importance of specific protein interaction domains.

These approaches can help elucidate how WDR83 orchestrates the assembly of signaling complexes and modulates signal transduction efficiency in various cellular contexts.

What are common issues when using WDR83 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with WDR83 antibodies. Here are solutions to common problems:

For optimal reproducibility, researchers should follow the specific storage and handling recommendations for their particular WDR83 antibody formulation.

How should researchers interpret WDR83 immunohistochemistry data in cancer tissues?

Interpreting WDR83 immunostaining in cancer tissues requires careful consideration of several factors:

Given WDR83's role in cancer pathophysiology, careful interpretation of its expression patterns may provide valuable insights into disease mechanisms and potential therapeutic targets.

How can researchers reconcile conflicting data when using different WDR83 antibodies?

When faced with conflicting results from different WDR83 antibodies, researchers should implement a systematic approach to resolve discrepancies:

• Epitope mapping analysis:

  • Compare the immunogen sequences of different antibodies

  • Antibodies targeting different domains may detect different isoforms or post-translationally modified forms of WDR83

  • Create a map of each antibody's target region relative to functional domains of WDR83

• Validation hierarchy implementation:

  • Prioritize data from antibodies that have undergone enhanced validation methods (genetic, orthogonal, recombinant expression validation)

  • Consider the extent of validation (e.g., Prestige antibodies validated across 44 tissue types and 20 cancer types)

  • Evaluate validation scores from repositories like the Human Protein Atlas

• Complementary technique confirmation:

  • Verify protein expression using mRNA detection methods (RT-PCR, RNAseq)

  • Consider mass spectrometry-based proteomics to confirm protein identity and abundance

  • Use genetic approaches (siRNA, CRISPR) to validate antibody specificity

• Cellular context considerations:

  • Different cellular states may affect epitope accessibility

  • WDR83's interaction with DHPS or components of MAPK pathways may mask certain epitopes

  • Consider fixation and sample preparation effects on epitope recognition

When publishing results, transparently report which antibody was used, including catalog number, lot number, and validation data to enable proper interpretation and reproducibility.

How can WDR83 antibodies be utilized to investigate its role in natural antisense transcript (NAT) regulation?

The discovery that WDR83 participates in natural antisense transcript regulation with DHPS opens exciting research avenues that can be explored using WDR83 antibodies:

• RNA-protein immunoprecipitation (RIP):

  • Use WDR83 antibodies to pull down associated RNA complexes

  • RT-PCR or RNA sequencing of precipitated material can identify RNA partners

  • This approach can reveal whether WDR83 protein directly interacts with DHPS mRNA or if intermediate proteins are involved

• Chromatin immunoprecipitation (ChIP):

  • Apply WDR83 antibodies in ChIP experiments to identify potential DNA binding sites

  • This can help determine if WDR83 plays a direct role in transcriptional regulation of sense/antisense pairs

• Subcellular co-localization studies:

  • Employ immunofluorescence with WDR83 antibodies (0.25-2 μg/mL) combined with RNA FISH for DHPS

  • This approach can reveal the cellular compartments where WDR83-DHPS interactions occur

• Dynamic regulation analysis:

  • Use WDR83 antibodies in time-course experiments following stimuli that affect NAT expression

  • Western blotting and immunoprecipitation at different time points can reveal the temporal dynamics of regulatory interactions

The bidirectional regulation between WDR83 and DHPS represents a novel regulatory mechanism with implications beyond this specific gene pair, potentially offering insights into broader antisense transcript regulatory networks .

What are the considerations for using WDR83 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence offers powerful insights into protein co-localization and pathway interactions, but requires careful optimization when incorporating WDR83 antibodies:

• Antibody compatibility assessment:

  • Test WDR83 antibodies with other target antibodies individually before multiplexing

  • Ensure host species compatibility to avoid cross-reactivity between secondary antibodies

  • For rabbit polyclonal WDR83 antibodies , pair with antibodies from different host species

• Fluorophore selection strategy:

  • Choose fluorophores with minimal spectral overlap

  • Consider the expected subcellular localization and expression level of WDR83

  • For potentially lower-expressed targets like WDR83, assign brighter fluorophores

• Protocol optimization:

  • Determine the optimal antibody concentration for immunofluorescence (0.25-2 μg/mL for some WDR83 antibodies)

  • Test sequential versus simultaneous antibody incubation approaches

  • Optimize antigen retrieval methods that work for all target proteins

• Validation approaches:

  • Include single-stain controls alongside multiplex experiments

  • Perform antibody stripping and reprobing experiments to confirm staining specificity

  • Consider computational approaches to correct for any spectral bleed-through

• Relevant multiplex combinations:

  • WDR83 with DHPS to study their co-localization and potential regulatory interactions

  • WDR83 with ERK pathway components to investigate its scaffold function

  • WDR83 with hypoxia markers to explore its role in hypoxia-inducible factor regulation

These considerations will help researchers develop robust multiplex immunofluorescence protocols that provide reliable insights into WDR83's biological functions and interactions.