IGHMBP2 Antibody

What is the optimal protocol for detecting IGHMBP2 by Western blot?

For reliable detection of IGHMBP2 by Western blot, follow these methodological steps:

• Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors.

• Protein separation: Load 20-40 μg of total protein per lane on 8-10% SDS-PAGE gels.

• Transfer: Use wet transfer at 100V for 90 minutes onto PVDF membrane.

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody: Incubate with anti-IGHMBP2 antibody (such as 23945-1-AP) at 1:1000-1:5000 dilution overnight at 4°C.

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG at 1:5000 dilution for 1 hour at room temperature.

• Detection: Develop using ECL reagent.

Expected results: The primary band should appear at approximately 109 kDa, corresponding to full-length IGHMBP2. A faster migrating band at ~60 kDa may occasionally be observed, which likely represents a proteolytic fragment of IGHMBP2 . Under stressful conditions, an additional band at ~130 kDa may also be detected .

What cell lines or tissues provide reliable positive controls for IGHMBP2 antibody validation?

Based on experimental validation data, the following samples serve as reliable positive controls:

IGHMBP2 shows variable expression across tissues, with measurable levels in multiple human tissues when compared to the housekeeping gene GAPDH . Fibroblasts and lymphoblastoid cell lines also express detectable levels of IGHMBP2 and can serve as additional positive controls .

How can I effectively visualize the subcellular localization of IGHMBP2?

IGHMBP2 shows both cytoplasmic and nuclear localization, with predominant expression in the cytoplasm. For optimal visualization of its subcellular distribution:

• Immunofluorescence protocol:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

  • Block with 5% BSA in PBS for 1 hour

  • Incubate with anti-IGHMBP2 antibody (1:100 dilution) overnight at 4°C

  • Use Alexa Fluor-conjugated secondary antibodies (1:500) for 1 hour at room temperature

  • Counterstain nucleus with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

• Co-localization markers:

  • For cytoplasmic fraction: Co-stain with ribosomal markers as IGHMBP2 associates with ribosomes

  • For nuclear fraction: Co-stain with transcription factors or DNA replication markers

The monoclonal antibody mAb11-24 has been experimentally validated for immunofluorescence detection of IGHMBP2 in human NT2 cells and mouse MN-1 cells, consistently showing predominantly cytoplasmic localization with a smaller nuclear fraction .

How do IGHMBP2 protein expression levels differ between normal and disease states?

IGHMBP2 protein levels show significant correlation with disease severity in neuromuscular disorders:

Western blot analysis of fibroblast and lymphoblastoid cell lines has demonstrated that IGHMBP2 protein levels correlate with clinical phenotype differences between CMT2 and SMARD1 . This suggests that residual IGHMBP2 activity may modify disease severity, making protein level quantification potentially valuable for prognosis.

What is the optimal methodology for co-immunoprecipitation of IGHMBP2 and its binding partners?

For successful co-immunoprecipitation of IGHMBP2 and its binding partners:

• Preparation of cell lysates:

  • Harvest cells (293T cells work well) at 80-90% confluence

  • Lyse in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, and protease inhibitors

  • Sonicate briefly and clear lysate by centrifugation at 14,000 × g for 15 minutes

• Immunoprecipitation procedure:

  • Pre-clear lysate with protein A/G beads for 1 hour at 4°C

  • Incubate pre-cleared lysate with anti-IGHMBP2 antibody (5 μg per 1 mg protein) overnight at 4°C

  • Add protein A/G beads and incubate for 3 hours at 4°C

  • Wash beads 4-5 times with wash buffer (same as lysis buffer but with 0.1% NP-40)

  • Elute by boiling in SDS sample buffer or use competitive elution with 3XFLAG peptide if using FLAG-tagged IGHMBP2

• Analysis of co-precipitated proteins:

  • Perform Western blot analysis for specific targets

  • Use mass spectrometry for unbiased identification of novel binding partners

Co-IP experiments have successfully demonstrated that IGHMBP2 interacts with Reptin (Tip48, RuvB-like2), Pontin (Tip49, RuvB-like1), and TFIIIC220 . IGHMBP2 has also been shown to self-associate, forming homooligomers in vivo . Interestingly, IGHMBP2 does not associate with components of the SMN complex (SMN, Gemin2, Gemin3, Gemin5, and Gemin6) .

How can I investigate IGHMBP2's association with ribosomes using antibody-based techniques?

To study IGHMBP2's association with ribosomes:

• Ribosome co-sedimentation assay:

  • Prepare cytoplasmic extract in buffer containing 20 mM HEPES pH 7.4, 100 mM KCl, 5 mM MgCl2, and 1 mM DTT

  • Layer extract on 10-50% sucrose gradient

  • Centrifuge at 35,000 rpm for 3 hours at 4°C

  • Collect fractions and analyze by Western blot for IGHMBP2 and ribosomal markers

  • Expected result: IGHMBP2 will co-fractionate with ribosomal subunits and intact ribosomes

• Immunoprecipitation of ribosomal proteins:

  • Perform IP using antibodies against ribosomal proteins

  • Probe for IGHMBP2 in the immunoprecipitates

  • Expected result: IGHMBP2 will be present in IPs of ribosomal proteins

• Proximity ligation assay:

  • Co-stain cells for IGHMBP2 and ribosomal proteins

  • Perform proximity ligation assay following manufacturer's protocol

  • Expected result: Positive signals indicating close proximity of IGHMBP2 to ribosomes

Research has demonstrated that IGHMBP2 associates with ribosomes and is likely functionally linked to translation . DSMA1-causing mutations in IGHMBP2 do not affect this ribosome binding but severely impair its ATPase and helicase activity .

What methodologies can be used to study IGHMBP2's interaction with tRNAs and other small RNAs?

To investigate IGHMBP2's interaction with small RNAs, particularly tRNAs:

• RNA immunoprecipitation (RIP):

  • Cross-link cells with 1% formaldehyde for 10 minutes

  • Lyse cells and sonicate to shear RNA

  • Immunoprecipitate IGHMBP2 using anti-IGHMBP2 antibody

  • Extract RNA from immunoprecipitates

  • Analyze by RT-PCR, northern blot, or RNA sequencing

• Northern blot analysis of co-precipitated RNAs:

  • Perform IP of IGHMBP2 from cell lysates

  • Extract RNA from immunoprecipitates

  • Separate RNA on denaturing polyacrylamide gels

  • Transfer to nylon membrane

  • Probe with specific oligonucleotide probes for tRNAs of interest

• End-labeling analysis:

  • Extract RNA from IGHMBP2 immunoprecipitates

  • 3'-end-label RNA with [32P]pCp and T4 RNA ligase

  • Analyze on denaturing polyacrylamide gels

  • Expected result: A strong band at ~75 nt corresponding to tRNAs

Experimental evidence has shown that IGHMBP2 associates with small RNAs, particularly tRNA Tyr. Northern blot analysis of RNAs isolated from IGHMBP2 immunoprecipitates has confirmed this association . The interaction with tRNAs may be relevant to IGHMBP2's role in translation and could be important for understanding its function in disease states.

How can I determine if disease-causing IGHMBP2 mutations affect its RNA binding capabilities?

To assess how disease-causing mutations affect IGHMBP2's RNA binding:

• Recombinant protein expression and purification:

  • Clone wild-type and mutant IGHMBP2 constructs into expression vectors

  • Express in E. coli or insect cells

  • Purify using affinity chromatography

  • Verify protein integrity by SDS-PAGE and Western blot

• RNA binding assays:

  • Electrophoretic Mobility Shift Assay (EMSA):

    • Incubate purified wild-type or mutant IGHMBP2 with radiolabeled RNA

    • Analyze complex formation on native polyacrylamide gels

    • Compare binding affinities between wild-type and mutant proteins

  • Filter binding assay:

    • Incubate increasing concentrations of protein with radiolabeled RNA

    • Filter through nitrocellulose membrane to retain protein-RNA complexes

    • Quantify bound RNA and calculate dissociation constants

• Functional assays:

  • ATP-dependent helicase assay:

    • Prepare double-stranded RNA substrates with one strand radiolabeled

    • Incubate with wild-type or mutant IGHMBP2 in the presence of ATP

    • Analyze unwinding activity by gel electrophoresis

    • Expected result: Disease-causing mutations in IGHMBP2 severely impair its ATPase and helicase activity

Research has shown that DSMA1-causing amino acid substitutions in IGHMBP2 severely impair its ATPase and helicase activity while not affecting ribosome binding . This suggests that the enzymatic activity of IGHMBP2 is critical for its function, and loss of this activity contributes to disease pathogenesis.

How can I measure changes in global translation caused by IGHMBP2 deficiency?

To quantify the effects of IGHMBP2 deficiency on global translation:

• Polysome profiling:

  • Prepare cytoplasmic extracts from control and IGHMBP2-deficient cells

  • Separate polysomes on 10-50% sucrose gradients

  • Monitor absorbance at 254 nm to generate polysome profiles

  • Expected result: IGHMBP2 deletion reduces the polysome:monosome ratio, indicating decreased translation

• Puromycin incorporation assay:

  • Treat cells with O-propargyl-puromycin (OPP)

  • Fix and permeabilize cells

  • Perform click chemistry to conjugate fluorophores to incorporated OPP

  • Analyze by flow cytometry or fluorescence microscopy

  • Expected result: IGHMBP2 deletion leads to decreased OPP incorporation, indicating reduced translation

• Metabolic labeling:

  • Pulse-label cells with 35S-methionine/cysteine

  • Prepare protein extracts and measure incorporated radioactivity

  • Analyze by SDS-PAGE and autoradiography

  • Expected result: Reduced incorporation in IGHMBP2-deficient cells

Experimental evidence has shown that IGHMBP2 deletion modestly reduces global translation as measured by polysome profiling and nascent protein synthesis assays . This supports the hypothesis that IGHMBP2 is functionally linked to translation, and that disruption of this function may contribute to disease pathogenesis.

What techniques can be used to investigate the activation of the integrated stress response (ISR) in IGHMBP2-deficient cells?

To study ISR activation in IGHMBP2-deficient cells:

• ATF4 reporter assays:

  • Generate cell lines expressing an ATF4 reporter construct (e.g., uORF1,2(ATF4)-mApple)

  • Measure reporter expression by flow cytometry or fluorescence microscopy

  • Compare IGHMBP2-deficient cells to control cells

  • Expected result: IGHMBP2 knockout cells demonstrate basal, chronic ISR activation

• Western blot analysis of ISR markers:

  • Prepare protein extracts from control and IGHMBP2-deficient cells

  • Perform Western blot analysis for:

    • Phosphorylated eIF2α (p-eIF2α)

    • ATF4

    • CHOP

    • Other ISR-regulated proteins

  • Expected result: Increased levels of these markers in IGHMBP2-deficient cells

• Pharmacological manipulation:

  • Treat cells with ISR inhibitors (e.g., ISRIB, GCN2iB)

  • Measure effects on ATF4 reporter expression or ISR marker levels

  • Expected result: Normalization of ISR activation in treated IGHMBP2-deficient cells

• Transcriptomic analysis:

  • Perform RNA-seq on control and IGHMBP2-deficient cells

  • Analyze differential expression of ISR-regulated genes

  • Expected result: Upregulation of ATF4 and other ISR-regulated genes in IGHMBP2-deficient cells

Research has demonstrated that IGHMBP2 knockout cells show basal, chronic activation of the integrated stress response, as evidenced by ATF4 upregulation . This suggests that ISR activation may be a key mechanism linking IGHMBP2 dysfunction to cellular pathology in diseases like SMARD1 and CMT2S.

How does the pattern of IGHMBP2 mutations correlate with different neuromuscular disease phenotypes?

IGHMBP2 mutations show distinct patterns that correlate with specific disease phenotypes:

Mutation analysis reveals:

• Truncating mutations in trans are consistently associated with SMARD1

• Non-truncating mutations in RecA-like domains (Domains 1A and 2A) are significantly more common in SMARD1 than in CMT2S (χ² = 6.893, p = 0.009)

• Most missense mutations causing SMARD1 are found in the helicase domain, indicating its critical role in disease pathogenesis

• Homozygous non-truncating mutations are more frequently associated with the milder CMT2S phenotype

These patterns suggest that IGHMBP2 protein levels and residual function determine the clinical phenotype, with higher levels and function resulting in milder disease.

What experimental approaches can distinguish pathogenic from non-pathogenic IGHMBP2 variants?

To differentiate pathogenic from non-pathogenic IGHMBP2 variants:

• Protein stability and expression analysis:

  • Transfect cells with wild-type or mutant IGHMBP2 constructs

  • Measure protein levels by Western blot

  • Assess protein stability using cycloheximide chase assay

  • Expected result: Pathogenic variants often show reduced stability and expression

• ATPase and helicase activity assays:

  • Express and purify recombinant wild-type and mutant IGHMBP2

  • Measure ATP hydrolysis using colorimetric assays

  • Assess helicase activity using fluorescence-based unwinding assays

  • Expected result: Pathogenic mutations severely impair ATPase and helicase activity while not affecting ribosome binding

• Cell-based functional assays:

  • Generate IGHMBP2-knockout cells complemented with wild-type or mutant IGHMBP2

  • Measure global translation using polysome profiling or OPP assays

  • Assess ISR activation using ATF4 reporter assays

  • Expected result: Pathogenic variants fail to rescue translation defects and ISR activation

• Clinical correlation:

  • Analyze IGHMBP2 protein levels in patient-derived cells

  • Compare with clinical severity

  • Expected result: Protein levels correlate with disease severity, with lower levels in more severe phenotypes

Research has shown that disease-causing mutations in IGHMBP2 severely impair its enzymatic activity . The c.2636C>A (p.T879K) variant, previously reported as pathogenic in the Human Gene Mutation Database, was found in both patients and healthy individuals, suggesting it may be a benign polymorphism . This highlights the importance of functional validation of IGHMBP2 variants.

How should I optimize immunostaining protocols for detecting IGHMBP2 in different tissue types?

For optimal IGHMBP2 immunostaining across different tissues:

• Tissue-specific fixation methods:

  • Neural tissues: Use 4% paraformaldehyde for 24 hours

  • Muscle tissues: Use 10% neutral buffered formalin for 24-48 hours

  • Preserve tissue morphology by processing and embedding in paraffin

• Antigen retrieval optimization:

  • For human liver cancer tissue: Use TE buffer pH 9.0 for heat-induced epitope retrieval

  • Alternative: Citrate buffer pH 6.0 may be used for other tissue types

  • Heat in pressure cooker or microwave until boiling, then 20 minutes at sub-boiling temperature

• Antibody dilution and incubation:

  • Primary antibody: Use anti-IGHMBP2 antibody at 1:20-1:200 dilution

  • Incubate overnight at 4°C in humid chamber

  • Secondary antibody: Use appropriate HRP-conjugated secondary antibody at 1:500 dilution

  • Incubate for 1 hour at room temperature

• Signal detection and amplification:

  • For low-expressing tissues: Use tyramide signal amplification system

  • For standard detection: Use DAB substrate

  • Counterstain with hematoxylin

  • Mount with permanent mounting medium

Immunohistochemistry for IGHMBP2 has been successfully performed on human liver cancer tissue using the 23945-1-AP antibody , while monoclonal antibody mAb11-24 has been validated for immunofluorescence in cultured cells .

What strategies can be employed when troubleshooting inconsistent IGHMBP2 detection in Western blots?

When facing inconsistent IGHMBP2 detection in Western blots:

• Sample preparation optimization:

  • Add phosphatase inhibitors alongside protease inhibitors

  • Include reducing agents (DTT or β-mercaptoethanol) in lysis buffer

  • Avoid multiple freeze-thaw cycles of protein samples

  • Process identical samples consistently to ensure reproducibility

• Band pattern analysis and verification:

  • Primary band: Expected at ~109 kDa (full-length IGHMBP2)

  • Degradation band: May appear at ~60-80 kDa (proteolytic fragment)

  • Extra band: May appear at ~130 kDa under stressful conditions

  • Verify specific bands by comparing patterns across multiple samples

• Antibody optimization:

  • Test multiple antibodies targeting different epitopes

  • Optimize antibody concentration (1:1000-1:5000 range for Western blot)

  • Consider longer primary antibody incubation (overnight at 4°C)

  • Verify antibody specificity using siRNA knockdown or IGHMBP2-knockout controls

• Technical considerations:

  • Ensure complete transfer of high molecular weight proteins

  • Use freshly prepared reagents and buffers

  • For problematic detection, try different membrane types (PVDF vs. nitrocellulose)

  • Consider using gradient gels for better resolution

Research has shown that Western blot results for IGHMBP2 can vary between sets of identical samples harvested and processed on different days , highlighting the importance of consistent sample handling. The existence of degradation bands around 70-80 kDa has been observed in individuals with CMT2 carrying specific mutations , which can complicate interpretation.

How can IGHMBP2 antibodies be used to distinguish between SMARD1 and other motor neuron diseases?

IGHMBP2 antibodies can be valuable tools for differentiating SMARD1 from other motor neuron diseases:

• Diagnostic immunohistochemistry protocol:

  • Collect muscle or nerve biopsy samples

  • Process and section tissues as described in Question 7.1

  • Perform immunostaining for IGHMBP2

  • Expected result: Reduced IGHMBP2 staining in SMARD1 patient tissues compared to controls and other motor neuron diseases

• Western blot analysis of patient-derived cells:

  • Isolate fibroblasts or lymphoblasts from patients

  • Prepare protein extracts and perform Western blot for IGHMBP2

  • Quantify IGHMBP2 protein levels relative to loading controls

  • Expected result: Significantly reduced IGHMBP2 levels in SMARD1 patients compared to controls and other motor neuron diseases

• Differential protein analysis:

  • Compare IGHMBP2 levels with SMN (Survival Motor Neuron) protein levels

  • Perform co-immunostaining for IGHMBP2 and SMN

  • Expected result: SMARD1 shows reduced IGHMBP2 but normal SMN, while SMA shows reduced SMN but normal IGHMBP2

• TDP43 pathology assessment:

  • Perform co-immunostaining for IGHMBP2 and TDP43

  • Expected result: TDP43 pathology may be observed in anterior horn cells of spinal cord in SMARD1 patients

Research has demonstrated that IGHMBP2 does not associate with the SMN complex , indicating distinct molecular pathways for SMARD1 compared to SMA despite clinical similarities. IGHMBP2 protein levels in fibroblasts and lymphoblasts are significantly higher in CMT2 than SMARD1, but lower than controls , providing a potential biochemical marker to distinguish these conditions.

What experimental approaches can assess the therapeutic potential of restoring IGHMBP2 function?

To evaluate therapeutic strategies aimed at restoring IGHMBP2 function:

• Gene replacement therapy assessment:

  • Transduce IGHMBP2-deficient cells with wild-type IGHMBP2 using viral vectors

  • Measure IGHMBP2 protein expression by Western blot

  • Assess functional rescue:

    • Global translation (polysome profiling, OPP assay)

    • ISR activation (ATF4 reporter, Western blot for ISR markers)

  • Expected result: Restoration of normal translation and reduction of ISR activation

• Small molecule screening:

  • Establish high-throughput screening assay using IGHMBP2 reporter cells

  • Screen compounds for:

    • Increased IGHMBP2 protein stability

    • Enhanced residual helicase activity of mutant IGHMBP2

    • Suppression of ISR activation

  • Validate hits in patient-derived cells

• Rescue experiments in disease models:

  • Express TagBFP-IGHMBP2 or IGHMBP2-TagBFP in IGHMBP2 KO cells

  • Assess restoration of normal cellular phenotypes:

    • Measure mApple/EGFP ratio in ATF4 reporter cells

    • Assess translation rates

  • Expected result: Transgenic IGHMBP2 expression normalizes cellular defects

• Combined therapeutic approaches:

  • Test ISRIB (integrated stress response inhibitor) in IGHMBP2-deficient cells

  • Assess effects on ATF4 reporter expression

  • Combine with IGHMBP2 gene replacement

  • Expected result: ISRIB reduces ISR activation in IGHMBP2 knockout cells