FEM1B Antibody, Biotin conjugated

What is FEM1B and why is it an important research target?

FEM1B (Fem-1 homolog B) is a 70 kDa protein belonging to the fem-1 family that functions as a critical component of an E3 ubiquitin-protein ligase complex. Its significance stems from multiple key cellular roles:

• Acts as a substrate recognition subunit within E3 ubiquitin ligase complexes

• Functions as a death receptor-associated protein mediating apoptosis

• Plays crucial roles in glucose homeostasis, particularly in pancreatic islets

• Serves as an adapter/mediator in replication stress-induced signaling leading to CHEK1 activation

These diverse functions make FEM1B antibodies valuable tools for investigating multiple cellular pathways including protein degradation, apoptosis, and metabolic regulation.

What applications are validated for FEM1B antibodies?

FEM1B antibodies, including biotin-conjugated variants, have been validated for multiple experimental applications with specific dilution recommendations:

For optimal results, it is recommended to titrate the antibody concentration for each specific experimental system and application.

What are the appropriate storage and handling conditions?

To maintain antibody integrity and activity:

• Store at -20°C for long-term preservation

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

• Stable for approximately one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations (20 μl sizes) contain 0.1% BSA

• Avoid repeated freeze-thaw cycles to prevent degradation

How should antigen retrieval be optimized for FEM1B detection in tissue samples?

Effective antigen retrieval is critical for successful FEM1B detection in fixed tissues. Based on experimental validation:

• Primary recommendation: Use TE buffer at pH 9.0 for optimal epitope accessibility

• Alternative method: Citrate buffer at pH 6.0 can be used with potentially different sensitivity profiles

• Buffer selection should be experimentally determined for specific tissue types

• Retrieval conditions should be optimized based on fixation method and duration

This methodological consideration is particularly important for pancreatic tissue samples, where FEM1B has demonstrated significant biological relevance.

What experimental controls are essential when using FEM1B antibodies?

Proper experimental controls are crucial for validating FEM1B antibody specificity:

• Positive controls: PC-3 cells, mouse pancreas tissue, and mouse testis tissue have demonstrated reliable FEM1B expression

• Knockout/knockdown validation: Several publications have utilized FEM1B knockout models or knockdown approaches to validate antibody specificity

• Peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

• Cross-reactivity testing: Verify species reactivity through comparative analysis (human, mouse, and rat reactivity has been confirmed for many FEM1B antibodies)

How can FEM1B antibodies be incorporated into multiplexed immunodetection protocols?

For co-localization studies examining FEM1B with other cellular markers:

• Compatible co-staining markers include glucagon, insulin, pancreatic polypeptide, and somatostatin for pancreatic studies

• Secondary antibody selection should consider host species (rabbit) to avoid cross-reactivity

• For multiplexed fluorescence applications, Cy2-conjugated secondary antibodies have demonstrated compatibility

• Biotin-conjugated FEM1B antibodies facilitate amplification strategies using streptavidin detection systems

• Sequential staining protocols may be necessary when using antibodies raised in the same host species

How can FEM1B antibodies be utilized to study protein degradation pathways?

FEM1B plays a critical role in targeted protein degradation (TPD) as a component of E3 ubiquitin ligase complexes. To study these pathways:

• Co-immunoprecipitation protocols using FEM1B antibodies can identify novel substrate interactions

• Combined with proteasome inhibitors (e.g., MG132), FEM1B antibodies can help elucidate degradation kinetics

• Proximity-based labeling approaches coupled with FEM1B antibody validation can map the degradation interactome

• When studying covalent FEM1B recruiters like EN106, antibodies can confirm target engagement

Recent research has demonstrated that FEM1B targets the C186 residue, which is critical for substrate recognition, making this region particularly important for degradation pathway studies.

What methodological approaches can investigate FEM1B's role in PROTAC development?

Proteolysis targeting chimeras (PROTACs) represent an emerging therapeutic modality leveraging E3 ligases for targeted protein degradation. When investigating FEM1B-recruiting PROTACs:

• Use FEM1B antibodies to validate expression levels in model cell lines before PROTAC testing

• Implement Western blot protocols with FEM1B antibodies to monitor FEM1B levels during PROTAC optimization

• Employ immunofluorescence to assess subcellular localization of FEM1B in relation to PROTAC targets

• Combine with ubiquitination assays to determine if FEM1B-recruiting PROTACs enhance ubiquitination of target proteins

The covalent FEM1B ligand EN106 has been successfully incorporated into PROTACs targeting BRD4 and BCR-ABL, demonstrating the utility of this approach for diverse protein targets .

How can researchers investigate the METTL3-FEM1B-GLI1 regulatory axis in skeletal stem cells?

Recent research has identified an important epitranscriptomic program involving the METTL3-FEM1B-GLI1 axis in maintaining skeletal stem cell homeostasis:

• Implement co-immunoprecipitation protocols using FEM1B antibodies to confirm interaction with GLI1

• Use cycloheximide (CHX) chase assays combined with FEM1B antibody detection to assess GLI1 protein stability

• Employ ubiquitination assays with FEM1B antibody validation to measure GLI1 polyubiquitination levels

• In Mettl3 knockout models, use FEM1B antibodies to monitor changes in FEM1B expression and subsequent effects on GLI1 levels

• For rescue experiments, validate FEM1B restoration using antibody detection methods

This experimental approach has revealed that FEM1B promotes GLI1 ubiquitylation and subsequent degradation, with m6A modification regulating FEM1B mRNA stability.

What techniques can assess FEM1B's role in glucose homeostasis and pancreatic islet function?

Given FEM1B's established role in glucose homeostasis, particularly in pancreatic islets:

• Use immunohistochemistry with FEM1B antibodies to characterize expression patterns in pancreatic β-cells and non-β-cells

• Implement co-localization studies with insulin and glucagon markers to determine cell type-specific expression

• In FEM1B knockout models, assess changes in glucose-stimulated insulin secretion

• Combine with arginine-stimulated insulin secretion assays to differentiate pathway-specific effects

• For mouse studies, established FEM1B-KO models have demonstrated abnormal glucose tolerance primarily due to defective glucose-stimulated insulin secretion

These methodological approaches have confirmed that FEM1B is expressed in both β-cells and non-β-cells within pancreatic islets and is highly expressed in INS-1E cells (a pancreatic β-cell line).

How should inconsistent results with FEM1B antibodies be addressed?

When encountering variability in FEM1B antibody performance:

• Verify antibody specificity using appropriate controls (knockout/knockdown systems where possible)

• Optimize protein extraction methods for different sample types (different lysis buffers may be required for preserving E3 ligase complex integrity)

• Adjust antigen retrieval conditions based on tissue type and fixation method

• Consider epitope accessibility issues, particularly when studying FEM1B in complex formations

• For detection of post-translational modifications, specialized extraction and preservation protocols may be necessary

What are the considerations when using FEM1B antibodies across different species?

While many FEM1B antibodies show cross-reactivity across species, important considerations include:

• Confirm reactivity for your specific species (well-validated for human, mouse, and rat samples)

• Compare sequence homology in the immunogen region for predicted cross-reactivity

• Use positive control tissues appropriate for each species (e.g., pancreas tissue for mice, PC-3 cells for human studies)

• Adjust antibody concentration when switching between species

• Be aware that some epitopes may be differently exposed or modified across species

Some antibodies show broader predicted reactivity including cow, sheep, horse, chicken, and rabbit, but experimental validation is recommended before extensive use in these species.