DCAF8 Antibody

What is DCAF8 and why are antibodies against it important for research?

DCAF8 (DDB1 and CUL4 associated factor 8) is a WD40 repeat-containing protein that functions as a substrate adapter for the Cullin-4 RING ubiquitin ligase complex (CRL4). This protein plays a critical role in targeted protein ubiquitination and subsequent proteasomal degradation . The significance of DCAF8 stems from its involvement in regulating protein stability for multiple substrates, including LSH (lymphoid-specific helicase) and MyHC (myosin heavy chain) .

Anti-DCAF8 antibodies are essential tools for investigating this protein's role in ubiquitination pathways, protein-protein interactions, and tissue-specific functions. These antibodies enable researchers to detect DCAF8 in various experimental contexts, including immunoblotting, immunoprecipitation, and immunohistochemistry applications . Without specific antibodies, studying DCAF8's expression patterns, interaction networks, and functional implications would be significantly more challenging.

What experimental applications are DCAF8 antibodies validated for?

DCAF8 antibodies have been validated for multiple experimental applications that are essential for comprehensive protein analysis:

• Western Blotting/Immunoblotting: Anti-DCAF8 antibodies have been successfully used for detecting endogenous DCAF8 in cell and tissue lysates at dilutions ranging from 1:500 to 1:1000 . These antibodies can identify DCAF8 species of approximately 70 kDa (matching the calculated molecular mass of ~67 kDa) and additional species at ~80 kDa that may represent post-translationally modified forms .

• Immunoprecipitation: DCAF8 antibodies have demonstrated effectiveness for immunoprecipitating endogenous DCAF8 and its interaction partners. Typical protocols use approximately 5 μg of antibody per immunoprecipitation reaction .

• Immunohistochemistry/Immunocytochemistry: These antibodies have been validated for detecting DCAF8 in fixed cells and tissues at dilutions of 1:50 to 1:100 .

• ELISA: While specific protocols aren't detailed in the provided sources, ELISA is listed as a common application for anti-DCAF8 antibodies .

When designing experiments with these antibodies, researchers should consider species cross-reactivity, as DCAF8 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

How can DCAF8 expression be accurately quantified using antibodies?

Accurate quantification of DCAF8 expression requires multiple complementary approaches:

Western Blot Quantification:

• Use validated anti-DCAF8 antibodies at manufacturer-recommended dilutions (typically 1:500-1:1000)

• Include appropriate loading controls such as β-actin, GAPDH, or vinculin antibodies for normalization

• Be aware that DCAF8 may appear as multiple bands (70 kDa and 80 kDa) representing different isoforms or post-translationally modified forms

• Use densitometry software to quantify band intensity relative to loading controls

Controls for Validation:

• Positive controls: Include tissues known to express DCAF8 (skeletal muscle, heart)

• Negative controls: Use DCAF8 knockout or knockdown samples as specificity controls

• Consider using siRNA-mediated DCAF8 depletion or CRISPR/Cas9-mediated DCAF8 ablation for antibody validation

When interpreting results, researchers should note that DCAF8 expression varies across tissues, with comparable levels detected in skeletal muscles like Gastrocnemius plantaris (GP) and Tibialis anterior (TA), heart, and other organs . Different tissues may also express tissue-specific isoforms, which could be detected as additional bands of various molecular weights (35 kDa and 50 kDa observed in pancreas, liver, and kidney) .

How can DCAF8 antibodies be optimized for co-immunoprecipitation of protein complexes?

Optimizing co-immunoprecipitation (co-IP) protocols for studying DCAF8 protein interactions requires careful consideration of several factors:

Buffer Optimization for Complex Stability:

• Use buffers that preserve native protein interactions while minimizing background

• For DCAF8 CRL4 complex studies, consider lysis buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease inhibitors

• Include phosphatase inhibitors when studying potential phosphorylated DCAF8 forms (~80 kDa species)

Antibody Selection and Validation:

• Validate antibody specificity using DCAF8 knockout or knockdown samples

• For co-IP of endogenous DCAF8 complexes, approximately 5 μg of anti-DCAF8 antibody per reaction has proven effective

• Consider using epitope-tagged DCAF8 constructs (FLAG, Myc, or HA tags) for pull-down experiments when appropriate

Detection of Complex Components:
When investigating DCAF8's role in CRL4 complexes, include antibodies against known components:

• Anti-DDB1 (1:500-1:1000 for immunoblotting)

• Anti-Cul4A (1:500)

• Anti-RBX1 (1:1000)

• Anti-MuRF1 (1:500)

Research has demonstrated that immunoprecipitation with anti-DCAF8 antibodies can successfully co-precipitate DDB1, Cul4A, RBX1, and MuRF1 from C2C12 myotube lysates, confirming DCAF8's participation in the CRL4 complex . Similarly, anti-DDB1 antibodies can co-precipitate Cul4A, DCAF8, RBX1, and MuRF1 . These findings highlight the stability and detectability of these protein interactions using optimized co-IP protocols.

What strategies should be employed when investigating DCAF8's role in muscle atrophy?

Investigating DCAF8's role in muscle atrophy requires multiple complementary approaches:

In Vitro C2C12 Myotube Atrophy Models:

• Differentiate C2C12 myoblasts into myotubes following established protocols

• Induce atrophy using dexamethasone (Dexa) treatment

• Quantify changes in myotube diameter as a measure of atrophy

• Manipulate DCAF8 expression through:

  • siRNA-mediated knockdown

  • CRISPR/Cas9-mediated knockout

  • Overexpression of wild-type or mutant DCAF8

Key Experimental Controls:

• Compare DCAF8 knockdown effects to MuRF1 knockdown (established atrophy mediator)

• Include combined DCAF8/MuRF1 knockdown conditions to assess potential synergistic effects

• Use scrambled siRNA controls for knockdown experiments

Protein Stability Assessments:

• Perform cycloheximide (CHX) chase experiments to determine the half-life of potential DCAF8 substrates

• Compare protein stability in control versus DCAF8 knockout/knockdown conditions

• Include proteasome inhibitors (e.g., MG132) to confirm the involvement of proteasomal degradation

Research has demonstrated that DCAF8 knockdown significantly impairs dexamethasone-induced decrease in myotube diameter in C2C12 cells, similar to the effects observed with MuRF1 knockdown . Combined downregulation of DCAF8 and MuRF1 prevented myotube shrinkage to approximately the same extent as single knockdowns, suggesting they may function in the same pathway . These findings support the concept that DCAF8 and MuRF1 form a functional unit that mediates muscle atrophy in cultured myotubes.

How can DCAF8-MuRF1 interactions be effectively studied and validated?

Investigating the interaction between DCAF8 and MuRF1 requires multiple orthogonal approaches:

Co-Immunoprecipitation Strategies:

• Immunoprecipitate endogenous DCAF8 using specific antibodies and probe for MuRF1 co-precipitation

• Perform reciprocal co-IP with anti-MuRF1 antibodies and detect DCAF8

• Use epitope-tagged constructs (DCAF8-FLAG, MuRF1-Myc) for co-IP in overexpression systems

Protein Stability Assessment:

• Determine whether DCAF8 and MuRF1 affect each other's stability using cycloheximide chase experiments

• Research indicates that DCAF8 and MuRF1 do not destabilize each other, suggesting a functional interaction rather than a substrate-enzyme relationship

Co-localization Studies:

• Perform immunofluorescence microscopy with anti-DCAF8 and anti-MuRF1 antibodies

• Use fluorescent protein-tagged constructs (RFP-DCAF8, GFP-MuRF1) to visualize localization in living cells

• Quantify co-localization using appropriate software and statistical analysis

Controls and Validation Approaches:

• Use DCAF8 knockout or knockdown cells to confirm antibody specificity

• Include unrelated protein controls in co-IP experiments

• Create point mutations in DCAF8's functional domains (e.g., WDXR motifs) to disrupt specific interactions

Research has revealed that MuRF1 and DCAF8 co-localize in cytoplasmic granular structures in COS-7 cells and in the cytosol of C2C12 cells . Furthermore, co-IP experiments have confirmed physical interaction between the proteins, but their interaction doesn't lead to mutual degradation . Interestingly, even in the absence of DCAF8 (using CRISPR/Cas9 knockout), MuRF1 can still interact with DDB1, suggesting multiple interaction modes within the CRL4 complex .

What are the best approaches for generating and validating DCAF8 knockout models?

Creating reliable DCAF8 knockout models requires careful design and thorough validation:

CRISPR/Cas9-Mediated DCAF8 Knockout:

• Design guide RNAs targeting early exons of DCAF8 (e.g., exon 2) using appropriate design tools

• Consider using a vector system that co-expresses a fluorescent marker (like mCherry-tagged Cas9) for cell sorting

• Clone annealed guide oligonucleotides into an appropriate CRISPR/Cas9 vector

• Transfect target cells and isolate single clones (e.g., through FACS sorting of fluorescent cells)

Guide RNA Design Example from Research:

• Guide sequence (including PAM): 5′-GCACCGTGGACAGCGCAAACGGG-3′

• Oligonucleotide 1: 5′-CACCGCACCGTGGACAGCGCAAAC-3′

• Oligonucleotide 2: 5′-AAACGTTTGCGCTGTCCACGGTGC-3′

Validation of DCAF8 Knockout:

• Genomic DNA validation:

  • Extract genomic DNA from isolated clones

  • PCR-amplify the targeted locus using appropriate primers

  • Example validation primers:

    • Forward: 5′-GCAAACCTGAAACCTGAGGC-3′

    • Reverse: 5′-GCTGTAGGCTCCTGGATGTG-3′

  • Sequence the amplicon to confirm mutations

• Protein-level validation:

  • Perform Western blotting with anti-DCAF8 antibodies

  • Ensure complete absence of DCAF8 protein at the expected molecular weight (70-80 kDa)

  • Check for potential truncated forms that might retain partial function

• Functional validation:

  • Assess known DCAF8-dependent processes

  • For muscle cells, examine resistance to atrophy induction

  • Analyze ubiquitination levels of known DCAF8 substrates

Research demonstrates that stable DCAF8 knockout C2C12 cells generated using this approach show altered protein interaction networks and resistance to atrophy stimuli, confirming both technical knockout success and biological relevance .

How do DCAF8 expression patterns vary across different tissues and what does this suggest about its function?

DCAF8 exhibits a complex expression pattern across tissues that provides insights into its biological roles:

Tissue Expression Profile:
Immunoblotting with anti-DCAF8 antibodies has revealed:

• Comparable expression levels in skeletal muscles (Gastrocnemius plantaris and Tibialis anterior)

• Similar expression in heart tissue

• Presence in various other organs

Multiple DCAF8 Forms:
Anti-DCAF8 antibodies detect distinct molecular weight species across tissues:

• A doublet of 70 kDa and 80 kDa bands in most tissues, with varying relative abundance

• Additional species of 35 kDa and 50 kDa specifically in pancreas, liver, and kidney

These diverse forms may represent:

• Tissue-specific isoforms (multiple DCAF8 isoforms are documented in protein databases)

• Post-translational modifications (phosphorylation sites are annotated for DCAF8)

• Products of limited proteolysis

When studying DCAF8 in a new tissue context, researchers should:

• Perform Western blots to characterize the specific forms present

• Consider potential post-translational modifications affecting function

• Investigate tissue-specific interaction partners that may direct DCAF8 activity

What methods can detect differential ubiquitination mediated by DCAF8?

Investigating DCAF8's role in protein ubiquitination requires specialized techniques:

In Vivo Ubiquitination Assays:

• Co-express DCAF8 with potential substrate proteins and HA/FLAG-tagged ubiquitin

• Treat cells with proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated proteins

• Immunoprecipitate the substrate of interest

• Probe immunoblots with anti-ubiquitin or anti-HA/FLAG antibodies to detect ubiquitination

• Include appropriate controls:

  • Unrelated DCAF factors (e.g., DDB2) to demonstrate specificity

  • DCAF8 mutants (e.g., DCAF8R314A) that disrupt DDB1 interaction

In Vitro Ubiquitination Systems:

• Immunopurify CRL4-DCAF8 complex components

• Combine with purified substrate, ubiquitin, E1, and E2 enzymes in an ATP-containing buffer

• Detect ubiquitination by immunoblotting

• This approach enables demonstration of direct ubiquitination in a molecularly defined reaction

Substrate Stability Assessments:

• Perform cycloheximide chase experiments in cells with normal, elevated, or reduced DCAF8 levels

• Monitor substrate half-life through Western blotting at various time points

• Combine with proteasome inhibitors to confirm the involvement of proteasomal degradation

Research has demonstrated that DCAF8 significantly promotes polyubiquitination of substrates like LSH, compared to control conditions or unrelated DCAF factors . Furthermore, mutations that disrupt DCAF8's interaction with DDB1 (DCAF8R314A) reduce substrate polyubiquitination . In vitro ubiquitination systems with immunopurified components have confirmed that CRL4-DCAF8 can directly catalyze substrate polyubiquitination .

How can antibodies help distinguish between DCAF8 isoforms and modified forms?

Differentiating between DCAF8 isoforms and post-translationally modified forms requires specialized approaches:

Antibody Selection Strategies:

• Use antibodies targeting different epitopes across the DCAF8 sequence:

  • N-terminal antibodies may detect all isoforms

  • C-terminal antibodies may miss truncated isoforms

  • Isoform-specific antibodies targeting unique regions where available

• Consider commercial antibodies with documented reactivity to specific isoforms:

  • Review manufacturer datasheets for isoform specificity

  • Some antibodies may recognize epitopes common to all isoforms (pan-DCAF8)

Distinguishing Post-translational Modifications:

• Phosphorylation analysis:

  • The 80 kDa DCAF8 form observed in C2C12 cells may represent phosphorylated DCAF8 (five putative phosphorylation sites are annotated)

  • Treat samples with phosphatase before immunoblotting to confirm phosphorylation

  • Use phospho-specific antibodies if available

• Other modifications:

  • Consider ubiquitination, SUMOylation, or other modifications that may alter DCAF8 mobility

  • Use modification-specific antibodies in combination with DCAF8 immunoprecipitation

Experimental Approaches:

• SDS-PAGE resolution optimization:

  • Use gradient gels (e.g., 4-12%) to maximize separation of different forms

  • Consider Phos-tag acrylamide for enhanced separation of phosphorylated species

• Two-dimensional gel electrophoresis:

  • Separate by isoelectric point and molecular weight

  • Especially useful for distinguishing phosphorylated forms

• Mass spectrometry analysis:

  • Immunoprecipitate DCAF8 and analyze by MS

  • Can identify specific modification sites and isoform-specific peptides

The research indicates that multiple DCAF8 species exist in various tissues, with a predominant doublet at 70-80 kDa and additional forms of 35 kDa and 50 kDa in specific tissues . Understanding which forms are present in your experimental system is crucial for accurate interpretation of DCAF8 functions.