FAM124B Antibody

What is FAM124B and what is its biological significance?

FAM124B (Family with sequence similarity 124B) is a protein that has been identified as a potential interaction partner of both CHD7 and CHD8 chromodomain helicase DNA binding proteins. CHD7 mutations are associated with CHARGE syndrome, an autosomal dominant multiple malformation disorder, while CHD8 is implicated in neurodevelopmental disorders (NDD) and autism spectrum disorders (ASD) . FAM124B is characterized as a mainly nuclear localized protein with widespread expression in embryonic and adult mouse tissues. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, researchers have concluded that Fam124B may play a role in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders .

What transcript variants of FAM124B exist and how do they differ?

In humans, two transcript variants of FAM124B have been identified:

• Transcript variant 1: Contains two exons with the ATG in exon one and the stop codon in exon 2, resulting in a protein with 455 amino acids (NP_001116251.1) .

• Transcript variant 2: Contains an alternate exon with an in-frame stop codon, leading to a shorter protein product with 272 amino acids (NP_079061.2) .

In mice, only one transcript homologous to the human transcript variant 1 has been described, containing 456 amino acids (NP_775601.1) .

What are the standard applications for FAM124B antibody?

FAM124B antibody has been validated for several standard research applications:

Note: It is recommended that this reagent should be titrated in each testing system to obtain optimal results .

How does FAM124B interact with CHD7 and CHD8 proteins?

The interaction between FAM124B and CHD7/CHD8 proteins has been characterized through several experimental approaches:

• SILAC with mass spectrometry: FAM124B was initially identified as a potential interaction partner of both CHD7 and CHD8 using stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry .

• Co-immunoprecipitation studies: Both transcript variants of FAM124B were confirmed to interact with CHD7 and CHD8 parts through co-immunoprecipitation experiments .

• Direct yeast two-hybrid experiments: These studies revealed that both transcripts of FAM124B directly interact with the CHD8 part containing amino acids 1789–2302 (NP_065971.2), while the FAM124B-CHD7 interaction appears to be indirect or the interacting area is outside of the CHD7 part spanning amino acids 1591-2181 (NP_060250.2) .

Since both FAM124B transcript variants share exon 1, researchers hypothesize that the FAM124B-CHD8 interacting area is located within exon 1 of FAM124B .

What is the subcellular localization of FAM124B and how can it be visualized?

FAM124B is primarily a nuclear-localized protein. This localization has been determined through several experimental approaches:

• Immunofluorescence staining: Using an anti-FAM124B antibody (ProteinTech, 21313-1-AP) on HeLa cells, both endogenous and overexpressed FAM124B was detected mainly in the nucleus .

• Nuclear fraction isolation: Western blot analysis of the nuclear cell fraction of untransfected HeLa cells using anti-FAM124B antibody revealed a specific band of approximately 57 kDa, supporting the nuclear localization of endogenous FAM124B .

For optimal visualization of FAM124B:

• Use fresh cells fixed with 4% paraformaldehyde for immunofluorescence

• For IHC on tissues, antigen retrieval with TE buffer pH 9.0 is suggested (alternatively, citrate buffer pH 6.0 may be used)

• For subcellular localization studies, counterstain with nuclear markers such as DAPI

How can siRNA be optimally used for FAM124B knockdown experiments?

For effective FAM124B knockdown experiments, the following approach has been validated:

siRNA sequences: The following FAM124B (Human) siRNA sequences have been used successfully:

• 5'-AUGUUCUAGAAAUGGAGUACUGACC-3'

• 5'-GCAGUUUAAGGUUCAAGAGAUCGGC-3'

• 5'-GGCUUGACCAUCAUAAAUUCUGAAC-3'

Transfection protocol:

• Seed approximately 3–4 × 10^4 cells per well in 8-well chamber slides

• When cells reach 70–90% confluence under conditions of 37°C and 5% CO₂, prepare transfection mixture

• Mix Lipofectamine 3000 reagent (0.15-0.3 μL) with Opti-MEM medium according to manufacturer's instructions

• Combine with 0.2 μg siRNA, 0.4 μL P3000 reagent, and 10 μL Opti-MEM medium in a 1:1 ratio

• Incubate the mixture at room temperature for 15 minutes

• Add the mixture to each well and incubate at 37°C and 5% CO₂ for 48 hours

• Perform analysis (e.g., immunofluorescence) 48 hours after siRNA treatment

To prevent cell proliferation from affecting migration assays, 5 μg/mL of mitomycin C can be added to the medium 2 hours before creating a scratch .

What is the tissue expression pattern of FAM124B and how is it best characterized?

FAM124B shows widespread expression in embryonic and adult mouse tissues. The expression pattern has been studied through multiple complementary techniques:

Techniques for expression analysis:

• Semiquantitative RT-PCR: Performed on RNA from wild-type CD1 mouse tissues and E9.5 and E12.5 embryos

• Quantitative real-time PCR (qRT-PCR): Validated the RT-PCR results using ΔCt values normalized to housekeeping genes (Gapdh, Hprt, and Sdna)

• Western blotting: Confirmed expression in adult mouse tissues

• Immunohistochemical staining (IHC): Used to visualize expression in tissue sections

• In situ hybridization (ISH): Performed with a full-length Fam124B RNA probe on brain sections

Key findings:

• FAM124B shows highest expression in lung and lowest in liver

• In the brain, FAM124B is highly expressed in the cortex, hippocampus subfields 1–3 (CA1-3), dentate gyrus, caudate putamen, and cerebellum

• There is overlapping expression between Chd7 and Fam124B at murine embryonic day E12.5

How can the specificity of FAM124B antibody be validated?

To validate the specificity of FAM124B antibody, multiple complementary approaches should be employed:

• Overexpression validation:

  • Transiently transfect cells (e.g., HeLa cells) with a plasmid expressing tagged FAM124B (e.g., FAM124B-1,3-pCMV-cmyc)

  • Perform immunofluorescence using both anti-FAM124B antibody and anti-tag antibody

  • Confirm colocalization of signals from both antibodies

• Western blot analysis:

  • Compare untransfected cell lysate (endogenous FAM124B) with lysate from cells overexpressing tagged FAM124B

  • Verify band sizes (approximately 51 kDa for overexpressed FAM124B-1 with tag, and approximately 57 kDa for endogenous FAM124B)

  • Include appropriate positive controls (K-562 cells show reliable detection)

• siRNA knockdown control:

  • Perform siRNA knockdown of FAM124B

  • Confirm reduction in antibody signal by immunofluorescence or Western blot

  • Compare with non-targeting siRNA control

• Blocking peptide competition assay:

  • Use a FAM124B recombinant protein antigen (such as NBP2-49625PEP) as a blocking peptide

  • Pre-incubate the antibody with the blocking peptide

  • Observe elimination or reduction of the specific signal in Western blot or immunostaining

What are the optimal conditions for detecting FAM124B in different experimental systems?

Optimal detection conditions vary by experimental system:

For Western Blot (WB):

• Recommended dilution: 1:500-1:1000

• Positive control: K-562 cells

• Expected molecular weight: 51 kDa (observed 57 kDa for endogenous protein due to potential post-translational modifications)

• Use nuclear fractions for enhanced detection of endogenous FAM124B

For Immunohistochemistry (IHC):

• Recommended dilution: 1:50-1:500

• Antigen retrieval: TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)

• Positive control tissues: Human colon cancer tissue

• Detection system: HRP-conjugated secondary antibody with DAB substrate recommended

For Immunofluorescence (IF):

• Fixation: 4% paraformaldehyde

• Permeabilization: 0.1% Triton X-100 in PBS

• Blocking: 5% BSA or normal serum

• Nuclear counterstain: DAPI recommended to confirm nuclear localization

What are the challenges in detecting endogenous versus overexpressed FAM124B?

Researchers face several challenges when detecting endogenous FAM124B compared to overexpressed protein:

Endogenous FAM124B detection challenges:

• Lower signal intensity: Endogenous FAM124B typically shows weaker signal in immunofluorescence compared to overexpressed protein

• Molecular weight discrepancy: Endogenous FAM124B appears at approximately 57 kDa versus the predicted 51 kDa, likely due to post-translational modifications

• Nuclear localization: Requires proper cellular fractionation for effective Western blot detection

• Tissue-specific expression levels: Expression varies significantly between tissues, with highest levels in lung and brain and lowest in liver

Overexpressed FAM124B detection advantages:

• Stronger signal: Provides robust detection in both immunofluorescence and Western blot

• Tag-based detection: Allows dual verification using both anti-FAM124B and anti-tag antibodies

• Expected molecular weight: Appears at the predicted size of approximately 51 kDa (plus tag size)

Recommendations for improving endogenous detection:

• Use nuclear fractionation for Western blot applications

• Optimize antigen retrieval methods for tissues

• Increase antibody concentration within recommended ranges

• Use signal amplification methods for immunofluorescence

• Select appropriate positive control tissues based on expression data

How does FAM124B expression vary during embryonic development?

Based on studies in mouse models, FAM124B shows specific expression patterns during embryonic development:

• Expression in early embryos: FAM124B expression has been documented in mouse embryos at stages E9.5 and E12.5

• Developmental significance: The overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 suggests a potential role in developmental processes related to CHARGE syndrome

• Brain development: High expression of Fam124B in the developing mouse brain indicates a possible role in neurodevelopmental processes, potentially relevant to autism spectrum disorders and other neurodevelopmental disorders

For studying developmental expression:

• In situ hybridization (ISH) with a full-length Fam124B RNA probe is effective for tissue localization

• Immunohistochemistry on embryonic tissue sections provides cellular-level resolution

• RT-PCR and qRT-PCR offer quantitative assessment of expression levels across developmental stages

What functional assays can be used to study FAM124B's biological role?

Several functional assays can be employed to investigate FAM124B's biological functions:

Cell migration assay:

• Seed 1 × 10^6 HUVECs in a six-well tissue culture plate with 2 ml of M199 medium (20% FBS)

• Add 5 µg/mL of mitomycin C to inhibit cell proliferation 2 hours before creating a scratch

• Create a controlled scratch wound using a P-200 pipette tip

• Perform FAM124B siRNA knockdown transfection

• Monitor wound closure over 24 hours

• Quantify the migrated area to assess FAM124B's role in cell migration

Protein interaction assays:

• Co-immunoprecipitation to confirm interactions with CHD7 and CHD8 in various cell types

• Yeast two-hybrid assays to map specific interaction domains

• Proximity ligation assay (PLA) to visualize protein-protein interactions in situ

Chromatin association studies:

• Chromatin immunoprecipitation (ChIP) to identify genomic regions associated with FAM124B

• ChIP-seq to map genome-wide binding patterns in relation to CHD7 and CHD8

CRISPR-Cas9 gene editing:

• Generate FAM124B knockout cell lines to study loss-of-function effects

• Create specific mutations to assess the functional importance of different protein domains

Transcriptional regulation analysis:

• RNA-seq following FAM124B knockdown or overexpression to identify regulated genes

• Quantitative PCR to validate expression changes in target genes

What is the potential role of FAM124B in disease pathogenesis?

Based on current research, FAM124B may be involved in multiple disease processes:

CHARGE syndrome:

• FAM124B interacts with CHD7, mutations in which cause CHARGE syndrome

• The overlapping expression pattern between Chd7 and Fam124B during embryonic development suggests a potential role in CHARGE syndrome pathogenesis

• FAM124B may influence CHD7-regulated developmental processes through its participation in chromatin remodeling complexes

Neurodevelopmental disorders (NDD) and autism spectrum disorders (ASD):

• FAM124B interacts with CHD8, which is implicated in NDD and ASD

• High expression of Fam124B in the developing mouse brain, particularly in regions relevant to cognition and behavior (cortex, hippocampus, cerebellum), supports a potential role in neurodevelopment

• FAM124B may participate in chromatin remodeling processes that regulate genes important for neuronal development and function

Research directions for disease mechanisms:

• Investigate FAM124B expression in patient-derived cells from individuals with CHARGE syndrome or ASD

• Assess genetic variations in FAM124B in patient cohorts

• Develop animal models with Fam124B mutations to study developmental and behavioral phenotypes

How might FAM124B function in chromatin remodeling complexes?

As an interaction partner of CHD7 and CHD8, FAM124B likely plays a role in chromatin remodeling processes:

Potential molecular functions:

• May serve as a scaffold protein that facilitates assembly of CHD7/CHD8-containing complexes

• Could modulate the chromatin remodeling activity of CHD7 and CHD8

• May recruit additional factors to CHD7/CHD8-containing complexes

• Could target these complexes to specific genomic regions

Experimental approaches to investigate chromatin-related functions:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify FAM124B binding sites

• RNA-seq following FAM124B manipulation to identify regulated genes

• In vitro nucleosome remodeling assays with purified components

• Mass spectrometry analysis of FAM124B-associated proteins in chromatin contexts

Nuclear localization significance:

• The primarily nuclear localization of FAM124B supports its potential role in chromatin-associated processes

• The interaction between FAM124B and CHD8 has been mapped to exon 1 of FAM124B, providing a foundation for structure-function studies

What are the differences in detecting and studying FAM124B transcript variants?

The two human FAM124B transcript variants present unique considerations for detection and functional analysis:

Structural differences:

• Transcript variant 1: 455 amino acids, contains two exons (NP_001116251.1)

• Transcript variant 2: 272 amino acids, contains an alternate exon with an in-frame stop codon (NP_079061.2)

• Both variants share exon 1, which contains the CHD8-interacting region

Detection considerations:

• Antibody epitope location: Antibodies targeting regions common to both variants will detect both forms, while those targeting unique regions will be variant-specific

• Molecular weight discrimination: Western blot can distinguish the variants based on size (approximately 51 kDa for variant 1 vs. 30 kDa for variant 2)

• Transcript-specific PCR: Design primers spanning unique exon junctions for selective amplification

Functional analysis approaches:

• Variant-specific overexpression: Clone and express each variant separately to assess functional differences

• Variant-selective knockdown: Design siRNAs targeting unique regions of each variant

• Domain mapping: Use truncation constructs to identify functional domains unique to each variant

Both transcript variants have been confirmed to interact with CHD7 and CHD8 parts through co-immunoprecipitation experiments, suggesting potentially overlapping functions .

What are the best practices for antibody validation in FAM124B research?

For rigorous FAM124B antibody validation, researchers should follow these best practices:

Multi-technique validation:

• Western blot analysis on endogenous and overexpressed protein

• Immunofluorescence with appropriate controls

• Immunohistochemistry on tissues with known expression patterns

• ELISA or other quantitative binding assays

Genetic controls:

• siRNA or shRNA knockdown to demonstrate specificity

• CRISPR-Cas9 knockout cells as negative controls

• Overexpression systems as positive controls

Epitope verification:

• Blocking peptide competition assays using recombinant FAM124B protein

• Multiple antibodies targeting different epitopes to confirm results

Cross-species validation:

• Test antibody reactivity across relevant species (human, mouse, rat)

• Verify sequence conservation at the epitope region

Documentation and reporting:

• Record all validation experiments in detail

• Report antibody catalog numbers, lot numbers, and dilutions

• Include all controls in publications

• Follow the antibody validation guidelines from the International Working Group for Antibody Validation (IWGAV)