APOBEC2 Antibody

What is APOBEC2 and in which tissues is it primarily expressed?

APOBEC2 (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 2) is a member of the APOBEC/AID (Activation Induced Deaminase) family of cytidine deaminases. Unlike other family members with established editing activities, APOBEC2 functions as a transcriptional repressor that binds chromatin and regulates gene expression .

APOBEC2 expression is largely restricted to striated muscle tissues, with particularly high expression in skeletal and cardiac muscle . Within muscle tissue, APOBEC2 shows differential expression patterns:

• Stronger expression in slow muscle fibers compared to fast fibers

• Higher abundance in soleus muscle than in gastrocnemius muscle

• Expression increases late during myoblast differentiation

What applications are APOBEC2 antibodies validated for?

APOBEC2 antibodies have been validated for several research applications as shown in the following table:

For optimal results, each antibody should be titrated in specific testing systems to obtain optimal results .

What dilutions are recommended for different applications of APOBEC2 antibodies?

Based on validated antibody products, the following dilutions are recommended:

Note that optimal dilutions are sample-dependent and should be determined experimentally for each specific application and antibody .

What antigen retrieval methods are most effective for APOBEC2 immunohistochemistry?

For optimal immunohistochemical detection of APOBEC2 in formalin-fixed, paraffin-embedded tissues, two effective antigen retrieval methods have been validated:

• Primary recommendation: TE buffer pH 9.0

  • This provides optimal epitope exposure while maintaining tissue morphology

• Alternative method: Citrate buffer pH 6.0

  • As demonstrated in validated protocols: "Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol"

The selection between these methods may depend on your specific tissue type and fixation conditions. For human skeletal muscle tissue, the citrate buffer method at pH 6.0 has been specifically validated and shown to produce clear staining patterns .

How should APOBEC2 antibodies be stored to maintain activity?

For optimal storage and maintained reactivity:

• Store at -20°C

• Most formulations remain stable for one year after shipment when stored properly

• Antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer

What controls should be used when working with APOBEC2 antibodies?

When designing experiments with APOBEC2 antibodies, include the following controls:

• Positive tissue controls:

  • Mouse/rat heart tissue

  • Mouse/rat skeletal muscle tissue

  • Human heart tissue

• Negative controls:

  • Non-muscle tissues (which express minimal APOBEC2)

  • APOBEC2 knockdown or knockout samples

  • Secondary antibody-only controls to assess non-specific binding

• Validation controls:

  • For knockdown studies, confirm APOBEC2 reduction by immunoblot

  • For ChIP experiments, include input controls to normalize enrichment

How can APOBEC2 antibodies be used to study muscle fiber type distribution?

APOBEC2 shows differential expression between slow and fast muscle fibers, making APOBEC2 antibodies valuable for muscle fiber typing studies:

• Immunohistochemical approach:

  • Use anti-APOBEC2 antibodies in serial sections alongside antibodies against different myosin HC isoforms

  • APOBEC2 staining is significantly stronger in slow myofibers compared to fast myofibers

  • This pattern can be used to assess fiber type distribution in normal and pathological conditions

• Quantification method:

  • Northern and Western blot analyses provide quantitative information on APOBEC2 abundance

  • APOBEC2 is more highly expressed in soleus compared to gastrocnemius muscle

  • This pattern is consistent in both young and old mice

• Deficiency impact:

  • In APOBEC2-deficient models, a shift in muscle fiber type distribution is observed

  • This can be analyzed using APOBEC2 antibodies alongside other muscle markers

How can APOBEC2 antibodies be effectively used in ChIP-Seq experiments?

APOBEC2 functions as a transcriptional repressor by binding to chromatin. ChIP-Seq experiments with APOBEC2 antibodies provide valuable insights into its genomic targets:

• ChIP-Seq protocol optimization:

  • Perform experiments in triplicate to ensure reproducibility

  • Only analyze peaks that appear in at least 2 out of 3 replicates

  • Calculate enrichment of APOBEC2 at specific loci over input using MACS2

• Time point selection:

  • Critical time points during differentiation should be analyzed

  • For C2C12 myoblast differentiation, 14 and 34 hours post-differentiation show significantly different APOBEC2 chromatin occupancy patterns

  • These time points precede major gene expression changes

• Data analysis approach:

  • Use DiffBind R package to compare ChIP-Seq signal at called peaks between time points

  • For C2C12 differentiation, 969 sites show differential APOBEC2 occupancy (FDR ≤ 0.05)

  • Most peaks (964/969) show increasing signal at later time points, with a mean fold change increase of 1.5

How should contradictory findings about APOBEC2 structure be addressed in antibody-based studies?

Research literature contains contradictory findings about APOBEC2 structure that may impact antibody-based studies:

• Crystal structure vs. solution structure discrepancy:

  • Crystal studies suggested APOBEC2 forms dimers

  • NMR solution structure reveals APOBEC2 is unambiguously monomeric in solution

  • The N-terminal tail (residues 1-40) that was removed for crystallization prevents dimerization in the full-length protein

• Experimental approach to resolve this contradiction:

  • Use antibodies that recognize different epitopes of APOBEC2

  • Compare full-length APOBEC2 and truncated APOBEC2 (41-224) binding

  • Assess native protein state through non-denaturing techniques prior to antibody application

  • Be cautious when interpreting structural models based on the crystal structure

• Impact on experimental design:

  • For co-immunoprecipitation studies, consider that APOBEC2 may not form homodimers

  • For functional studies, the monomeric state of the protein may be physiologically relevant

  • When developing new antibodies, epitopes in the N-terminal region may affect recognition of the native protein

How can APOBEC2 antibodies be used to study muscle differentiation mechanisms?

APOBEC2 plays a key role in muscle differentiation by repressing non-muscle genes. Antibody-based approaches can elucidate these mechanisms:

• Combined ChIP-Seq and RNA-Seq approach:

  • Perform ChIP-Seq with APOBEC2 antibodies at early differentiation time points

  • Follow with RNA-Seq at later time points to correlate binding with expression changes

  • APOBEC2 occupancy at promoters correlates with repression of non-muscle lineage genes

• Analysis of APOBEC2 knockdown effects:

  • Compare transcriptomes of APOBEC2 knockdown and control cells during differentiation

  • Genes downregulated during normal differentiation are enriched for muscle development GO terms

  • Genes upregulated with APOBEC2 deficiency relate to non-muscle lineages (immune system, blood vessel, nervous system development)

• Motif analysis for APOBEC2 binding:

  • Annotate APOBEC2 ChIP-Seq peaks by genomic feature (most fall within promoter regions)

  • Assess motif signatures through enrichment analysis of transcription factor 8-mer sequences

  • This reveals APOBEC2's role in binding specific motifs within promoter regions

What molecular weight should be detected when using APOBEC2 antibodies in Western blot?

When performing Western blot with APOBEC2 antibodies, researchers should expect:

• Calculated molecular weight: 26 kDa

• Observed molecular weight range: 25-30 kDa

If bands appear at significantly different sizes, consider the following:

• Post-translational modifications may affect migration

• Sample preparation conditions (reducing vs. non-reducing) can impact apparent molecular weight

• Antibody specificity should be verified using positive and negative controls

How should researchers interpret differences in APOBEC2 antibody reactivity across species?

APOBEC2 antibodies show different cross-reactivity patterns across species:

When working with species not explicitly validated:

• Perform preliminary validation experiments

• Compare sequence homology in the epitope region

• Include appropriate positive and negative controls

What factors can affect APOBEC2 antibody performance in ChIP experiments?

When using APOBEC2 antibodies for chromatin immunoprecipitation:

• Critical factors affecting performance:

  • Fixation conditions significantly impact chromatin accessibility and epitope exposure

  • APOBEC2 expression levels vary dramatically during differentiation, affecting signal strength

  • Background binding can obscure true signals, requiring careful input normalization

• Optimization approaches:

  • Test multiple antibody concentrations (typically 0.5-4.0 μg per experiment)

  • Compare results from at least two different APOBEC2 antibodies recognizing distinct epitopes

  • Include APOBEC2 knockdown controls to confirm signal specificity

  • Analyze data using both peak calling (MACS2) and differential binding (DiffBind) approaches