RANBP2 Antibody

What is RANBP2 and what are its key functional roles?

RANBP2, also known as Nucleoporin 358 (Nup358), is a large 358 kDa protein that functions as a component of the nuclear pore complex. It serves as an E3 SUMO-protein ligase facilitating SUMO1 and SUMO2 conjugation by UBE2I. RANBP2 is involved in transport factor (Ran-GTP, karyopherin)-mediated protein import via its F-G repeat-containing domain, which acts as a docking site for substrates. It also functions as a component of the nuclear export pathway, providing a specific docking site for the nuclear export factor exportin-1. Additionally, RANBP2 inhibits EIF4E-dependent mRNA export and can sumoylate PML at 'Lys-490', which is essential for proper assembly of PML nuclear bodies. It also recruits BICD2 to the nuclear envelope during G2 phase and is considered a probable inactive PPIase with no peptidyl-prolyl cis-trans isomerase activity .

Which RANBP2 antibodies are currently available for research purposes?

Several RANBP2 antibodies are available for research applications, including:

• Rabbit polyclonal antibody ab64276 from Abcam, suitable for IP, WB, and ICC/IF applications with human samples

• Rabbit polyclonal antibody 27606-1-AP from Proteintech, which can be used in WB, IHC, and ELISA applications with human, mouse, and rat samples

• Other reported antibodies include A301-796A, sc-74518, ABN1385, and ab2938, each recognizing different regions of RANBP2

How should researchers determine the optimal RANBP2 antibody for their specific application?

When selecting a RANBP2 antibody, researchers should consider:

• Intended application (Western blot, immunohistochemistry, immunofluorescence)

• Species reactivity relevant to your experimental system

• Epitope recognition site within RANBP2 (different antibodies target different regions)

• Validation status (KO/KD validated antibodies are preferable)

• Published literature using the antibody for similar applications

Most importantly, researchers should thoroughly validate any antibody in their specific experimental system using appropriate controls such as RANBP2 knockdown/knockout samples. Pay special attention to antibody specificity concerns, as some RANBP2 antibodies have been shown to cross-react with other proteins such as neurofascin .

How can I validate the specificity of my RANBP2 antibody?

Thorough validation of RANBP2 antibodies is critical due to documented specificity issues. A comprehensive validation approach should include:

• Testing multiple independent antibodies recognizing different epitopes of RANBP2

• Using knockdown or knockout controls to verify signal reduction/elimination

• Performing immunoblotting to confirm expected molecular weight (358 kDa)

• Conducting epitope mapping experiments when cross-reactivity is suspected

• CRISPR/Cas9 epitope tagging of endogenous RANBP2 as an alternative approach

What are the known cross-reactivity issues with RANBP2 antibodies?

A significant cross-reactivity issue has been documented with the anti-RANBP2 antibody A301-796A. This antibody recognizes the amino acid sequence KPLQG, which is present in both RANBP2 and neurofascin (a well-established axon initial segment protein). This cross-reactivity led to misinterpretation of RANBP2 localization in neurons. Other antibodies that recognize different regions of RANBP2 (ABN1385 detecting amino acids 2033-3088 and sc-74518 detecting amino acids 1-1047) do not show this cross-reactivity pattern. When selecting antibodies, researchers should be aware of these specificity issues and use multiple antibodies or genetic validation approaches to confirm their findings .

How do different RANBP2 antibodies compare in terms of epitope recognition?

Different commercially available RANBP2 antibodies target distinct regions of this large protein:

This variation in epitope recognition is important to consider when designing experiments, as different regions of RANBP2 may be accessible depending on the experimental conditions and protein conformation .

What are the optimal experimental conditions for RANBP2 antibody-based Western blotting?

For optimal Western blotting with RANBP2 antibodies, consider the following protocol elements:

• Sample preparation: Given RANBP2's large size (358 kDa), use low percentage (6-7%) SDS-PAGE gels or gradient gels

• Transfer conditions: Extended transfer times (2-3 hours) or overnight transfer at low voltage is recommended for large proteins

• Blocking: 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature

• Primary antibody: For 27606-1-AP, use dilutions of 1:500-1:2000; for ab64276, follow manufacturer recommendations

• Incubation: Overnight at 4°C for best results

• Detection: Use high-sensitivity detection systems due to potential low expression levels

• Positive controls: Include HeLa cell lysate, which has been verified to express RANBP2

The high molecular weight of RANBP2 requires special attention to ensure complete transfer to the membrane. Using appropriate molecular weight markers and positive controls is essential for accurate identification .

What are the recommended protocols for immunohistochemistry with RANBP2 antibodies?

For immunohistochemistry using RANBP2 antibodies, follow these guidelines:

• Fixation: 4% paraformaldehyde is generally suitable

• Antigen retrieval: For antibody 27606-1-AP, use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• Antibody dilution: For 27606-1-AP, use 1:500-1:2000 dilution

• Incubation time: Overnight at 4°C for primary antibody

• Detection system: Use highly sensitive detection methods appropriate for your tissue

• Positive controls: Mouse kidney tissue and rat liver tissue have been verified for 27606-1-AP

• Negative controls: Include secondary antibody-only controls and, ideally, RANBP2 knockdown tissues

When interpreting results, be aware that RANBP2 typically shows nuclear membrane localization, consistent with its role as a nuclear pore complex component. Claims of significant cytoplasmic or specialized subcellular localization should be thoroughly validated with multiple antibodies and genetic approaches .

How should I approach subcellular localization studies for RANBP2?

When studying RANBP2 subcellular localization, implement a multi-faceted approach:

• Use multiple independent antibodies targeting different RANBP2 epitopes

• Include co-staining with established nuclear pore complex markers

• Complement antibody-based detection with genetic tagging approaches

• Consider CRISPR/Cas9-mediated endogenous tagging as demonstrated in the literature

• Implement proper controls to rule out cross-reactivity issues

The literature shows that endogenously expressed RANBP2 localizes primarily at the nuclear membrane, consistent with its function as a nucleoporin. Previous reports of RANBP2 at the axon initial segment have been disproven through careful antibody cross-reactivity analysis. When exogenously expressing RANBP2 fragments, be aware that their localization may not reflect endogenous RANBP2 behavior. For example, an N-terminal fragment of RANBP2 (amino acids 2-1047) can localize to the axon initial segment when overexpressed, likely due to its interaction with stabilized microtubules, but this does not reflect normal endogenous RANBP2 distribution .

Why might I observe multiple bands when performing Western blot for RANBP2?

Multiple bands in RANBP2 Western blots may occur for several reasons:

• Proteolytic degradation: RANBP2 is a large protein susceptible to degradation during sample preparation

• Alternative splicing: Multiple isoforms may exist in certain tissues

• Post-translational modifications: SUMO modifications or phosphorylation can alter migration

• Cross-reactivity: Some antibodies may recognize other proteins, as seen with the A301-796A antibody

• Incomplete denaturation: Large proteins may not fully denature, resulting in aberrant migration

To address these issues:

• Include protease inhibitors in all lysis buffers

• Freshly prepare samples and avoid freeze-thaw cycles

• Use appropriate positive controls

• Test antibody specificity in knockout/knockdown systems

• Compare results with multiple antibodies targeting different epitopes

The antibody A301-796A has been shown to recognize both the 358 kDa RANBP2 and ~190 and ~150 kDa proteins (later identified as neurofascin) in brain homogenates. Understanding these patterns is crucial for accurate interpretation of results .

How can I address weak or absent RANBP2 signal in my experiments?

If you encounter weak or absent RANBP2 signal, consider these approaches:

• Antibody concentration: Titrate antibody to find optimal concentration; 1:500-1:2000 is recommended for 27606-1-AP

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C

• Detection sensitivity: Use high-sensitivity ECL substrates for Western blot

• Protein loading: Increase total protein amount loaded (particularly for tissues with lower RANBP2 expression)

• Epitope accessibility: Try different antibodies targeting distinct epitopes

• Antigen retrieval: For IHC, optimize antigen retrieval methods (TE buffer pH 9.0 recommended for 27606-1-AP)

• Sample preparation: Ensure efficient extraction of nuclear membrane proteins

For large proteins like RANBP2, extended transfer times are crucial. Additionally, avoid harsh detergents that might destroy the epitope structure. If problems persist, consider alternative antibodies or complementary approaches such as mRNA detection .

How do I interpret conflicting localization results between different RANBP2 antibodies?

When facing conflicting localization results with different RANBP2 antibodies:

• Evaluate antibody specificity: Test each antibody in knockout/knockdown systems

• Consider cross-reactivity: Check if antibodies might recognize other proteins

• Map epitope recognition: Determine which domain of RANBP2 each antibody recognizes

• Assess accessibility: Some epitopes may be masked in certain conformations or contexts

• Compare with genetic approaches: Use CRISPR/Cas9 epitope tagging as an antibody-independent method

• Consider functional evidence: Align localization claims with known RANBP2 functions

How can RANBP2 antibodies be used to study its role in protein transport and SUMO-conjugation?

To investigate RANBP2's roles in protein transport and SUMO-conjugation:

• Co-immunoprecipitation using RANBP2 antibodies to identify interaction partners

• Proximity labeling approaches (BioID, APEX) coupled with RANBP2 antibody validation

• Super-resolution microscopy to visualize RANBP2 at nuclear pores using validated antibodies

• In vitro SUMO conjugation assays with immunoprecipitated RANBP2

• ChIP-seq approaches to identify potential DNA binding sites

When designing these experiments, be mindful of RANBP2's multiple domains and activities. Different regions of the protein are responsible for specific functions: the N-terminal region contains the leucine-rich region and interacts with SUMO1, the central region contains zinc finger motifs and RanGTP binding domains, and the C-terminal region contains cyclophilin-like domain. Choose antibodies targeting relevant domains based on your specific research questions .

What is the significance of RANBP2 in disease contexts, and how can antibodies facilitate this research?

RANBP2 has been implicated in several disease contexts, and antibodies can facilitate this research through:

• Expression analysis in patient samples: Some studies suggest RANBP2 upregulation in early multiple myeloma development (MGUS) and unfavorable disease conditions

• Localization studies in diseased tissues: Changes in RANBP2 distribution may correlate with pathology

• Post-translational modification analysis: Alterations in SUMO-conjugation activity may contribute to disease

• Interaction studies: Changes in RANBP2's interaction network may provide disease insights

How can CRISPR/Cas9 epitope tagging approaches complement antibody-based RANBP2 research?

CRISPR/Cas9 epitope tagging offers powerful complementary approaches to antibody-based RANBP2 research:

• Endogenous tagging strategy: Insert small epitope tags (HA, FLAG) at N-terminal, C-terminal, or internal sites of RANBP2

• sgRNA design: Target specific exons based on tag insertion location (e.g., exon 1 for N-terminal tagging)

• Tag detection: Use well-validated commercial antibodies against epitope tags

• Verification: Confirm successful tagging through genomic PCR and sequencing

• Functional assessment: Ensure tagged RANBP2 retains normal functionality

This approach provides several advantages:

• Avoids potential cross-reactivity issues of RANBP2 antibodies

• Enables tracking of endogenous protein without overexpression artifacts

• Allows for clean immunoprecipitation experiments

• Facilitates live-cell imaging when combined with fluorescent tags

As demonstrated in the literature, researchers have successfully used CRISPR/Cas9 to insert HA-epitope tags at various positions in the endogenous RANBP2 gene, confirming its nuclear membrane localization and resolving conflicts arising from antibody cross-reactivity issues .

What are the current limitations in RANBP2 antibody research that need to be addressed?

Current limitations in RANBP2 antibody research include:

• Specificity issues with certain commercial antibodies

• Limited validation in diverse experimental systems and tissues

• Challenges in detecting the full-length 358 kDa protein

• Incomplete characterization of epitope accessibility in different cellular contexts

• Potential isoform-specific detection challenges

The case of A301-796A antibody cross-reactivity with neurofascin serves as an important reminder of the need for rigorous validation through multiple approaches. Future antibody development should focus on isoform-specific reagents and broader validation across diverse experimental systems .

What emerging methodologies might enhance RANBP2 research beyond traditional antibody approaches?

Emerging methodologies that could advance RANBP2 research include:

• Nanobody development against specific RANBP2 domains

• Targeted protein degradation approaches (PROTACs, dTAG) to study RANBP2 functions

• Mass spectrometry-based approaches for label-free quantification

• Advanced imaging techniques like super-resolution microscopy combined with genetic tagging

• Single-cell proteomics to assess RANBP2 expression heterogeneity

• Structural biology approaches (Cryo-EM) to understand RANBP2's complex architecture