FAM222B Antibody

What is FAM222B protein and what are the available antibody types for its detection?

FAM222B (also known as C17orf63, chromosome 17 open reading frame 63, FLJ10700, or hypothetical protein LOC55731) is a human protein localized primarily in the nucleoplasm . For its detection, researchers can use several types of antibodies:

• Rabbit polyclonal antibodies (most common)

• Host-specific antibodies conjugated with detection tags such as HRP

• Affinity-purified antibodies for enhanced specificity

These antibodies are typically raised against recombinant fragments of human FAM222B protein, with immunogens corresponding to different amino acid regions such as aa 100-200 or aa 351-562 .

What are the validated applications for FAM222B antibodies?

FAM222B antibodies have been validated for multiple experimental applications:

Experimental validation shows positive detection in multiple human cell lines and tissues including RT-4, U-251 MG cell lysates, human plasma, liver, tonsil, and rectum tissues .

How should I optimize FAM222B antibody concentration for different applications?

Optimization is critical for obtaining specific signals while minimizing background. For FAM222B antibodies:

Western Blot Optimization:

• Start with the manufacturer's recommended dilution (typically 1:250)

• Perform a dilution series (e.g., 1:100, 1:250, 1:500, 1:1000)

• Include appropriate positive controls (e.g., RT-4 or U-251 MG cell lysates)

• Verify the band size (predicted molecular weight is approximately 60 kDa)

• Adjust blocking conditions (5% non-fat milk or BSA) if non-specific binding occurs

Immunohistochemistry/Immunocytochemistry Optimization:

• Begin with recommended dilutions (1:50 for IHC-P, 1-4 μg/ml for ICC/IF)

• Test antigen retrieval methods (heat-induced vs. enzymatic)

• Adjust incubation time and temperature

• Include positive control tissues (e.g., human rectum for IHC-P)

ELISA Optimization:

• Start with 1:500 dilution and adjust based on signal intensity

• Test different coating concentrations of capture antigen

• Optimize secondary antibody dilution

• Determine optimal substrate incubation time

What are the recommended sample preparation techniques for optimal FAM222B detection?

Sample preparation significantly impacts antibody performance:

For Western Blot:

• Use RIPA or NP-40 buffer with protease inhibitors for cell/tissue lysis

• Determine optimal protein loading (typically 10-30 μg per lane)

• Ensure complete protein denaturation (boil in Laemmli buffer for 5 minutes)

• Include phosphatase inhibitors if phosphorylation status is relevant

For IHC-P:

• Fix tissues in 10% neutral buffered formalin (24-48 hours)

• Use proper paraffin embedding and sectioning techniques (4-6 μm sections)

• Test different antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Block endogenous peroxidase activity (3% H₂O₂) and non-specific binding sites

For ICC/IF:

• Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 (5-10 minutes)

• Block with appropriate serum (5-10% normal serum from the same species as secondary antibody)

• Include nuclear counterstain (e.g., DAPI) to verify nuclear localization

How can I verify the specificity of FAM222B antibody in my experimental system?

Antibody specificity is critical for reliable results. Verify FAM222B antibody specificity through:

• Positive and Negative Controls:

  • Include known positive controls (e.g., human cell lines with documented FAM222B expression)

  • Use negative controls (tissues/cells known to lack FAM222B expression)

  • Apply isotype controls to rule out non-specific binding

• Knockdown/Knockout Validation:

  • Compare staining in wild-type vs. FAM222B-knockdown/knockout samples

  • Verify signal reduction/elimination in depleted samples

• Peptide Competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Observe signal reduction/elimination in peptide-blocked samples

• Orthogonal Detection Methods:

  • Confirm protein expression using alternative antibodies targeting different epitopes

  • Correlate protein expression with mRNA levels (RT-PCR, RNA-seq)

• Specificity Testing:

  • Some FAM222B antibodies have verified specificity on protein arrays containing the target protein plus 383 other non-specific proteins

Does FAM222B antibody show species cross-reactivity, and how can I determine this?

Based on available data, most commercial FAM222B antibodies are validated for human samples . To determine potential cross-reactivity:

• Sequence Homology Analysis:

  • Compare FAM222B protein sequences across species

  • Identify the epitope region recognized by the antibody

  • Calculate percent identity in the epitope region

• Empirical Cross-Reactivity Testing:

  • Test antibody on samples from multiple species

  • Use tissues with known FAM222B expression patterns

  • Compare staining patterns and band sizes across species

• Manufacturer Information:

  • Check product datasheets for verified cross-reactivity

  • Contact manufacturers for unpublished cross-reactivity data

• Literature Review:

  • Search for publications using the specific antibody across species

When cross-reactivity data is limited, consider using species-specific antibodies rather than assuming cross-reactivity based on sequence homology alone.

How can I optimize FAM222B antibody for multiplexed immunofluorescence studies?

Multiplexed immunofluorescence allows simultaneous detection of multiple proteins:

• Antibody Panel Design:

  • Select FAM222B antibody with validated performance in immunofluorescence

  • Ensure primary antibodies are from different host species or different isotypes

  • Choose complementary fluorophores with minimal spectral overlap

• Sequential Staining Protocol:

  • Start with lowest concentration antibody first

  • Use tyramide signal amplification for weak signals

  • Include proper washing steps between antibody applications

  • Consider microwave treatment for antibody stripping if using same-species antibodies

• Controls for Multiplexing:

  • Single-stain controls to verify specificity and rule out bleed-through

  • Fluorophore-minus-one controls to establish proper compensation

  • Blocking between sequential rounds to prevent cross-reactivity

• Imaging Considerations:

  • Optimize exposure settings for each fluorophore

  • Perform linear unmixing for overlapping spectra

  • Use appropriate filters to minimize bleed-through

FAM222B has been observed in the nucleus but excluded from nucleoli in human CACO-2 cells , making it suitable for co-localization studies with other nuclear or nucleolar markers.

What are the considerations for using FAM222B antibody in chromatin immunoprecipitation (ChIP) studies?

While the search results don't specifically mention ChIP applications for FAM222B antibodies, researchers interested in exploring FAM222B interactions with chromatin should consider:

• Antibody Selection:

  • Choose antibodies validated for immunoprecipitation

  • Ensure the epitope is accessible in cross-linked chromatin

  • Verify antibody specificity in IP conditions

• Protocol Optimization:

  • Test different cross-linking conditions (0.5-2% formaldehyde, 5-15 minutes)

  • Optimize sonication parameters for appropriate chromatin fragment size (200-500 bp)

  • Determine optimal antibody concentration and incubation conditions

  • Include appropriate positive controls (e.g., histone marks) and negative controls (IgG)

• Validation Approaches:

  • Perform Western blot on input and IP samples to confirm enrichment

  • Include known positive and negative genomic regions in qPCR validation

  • Consider ChIP-seq to identify genome-wide binding patterns

• Data Analysis Considerations:

  • Use appropriate normalization methods (percent input, IgG control)

  • Perform biological replicates to ensure reproducibility

  • Correlate ChIP data with expression or functional data

Since FAM222B localizes to the nucleoplasm , ChIP studies could provide valuable insights into its potential role in gene regulation.

What are common issues in FAM222B antibody-based assays and how can they be resolved?

How should I interpret and validate unexpected FAM222B expression patterns or subcellular localizations?

When encountering unexpected results:

• Verify with Multiple Approaches:

  • Use different antibodies targeting distinct epitopes

  • Compare results across multiple detection methods (WB, IHC, ICC, IF)

  • Correlate protein expression with mRNA levels

• Consider Biological Variables:

  • Cell type-specific expression patterns

  • Effects of cell cycle stage on localization

  • Stimulus-dependent translocation (e.g., stress, signaling)

  • Post-translational modifications affecting epitope recognition

• Technical Validation:

  • Test different fixation and permeabilization methods

  • Compare fresh vs. frozen vs. FFPE samples

  • Control for autofluorescence or endogenous peroxidase activity

• Literature Comparison:

  • Compare results with published FAM222B localization (nucleoplasm but excluded from nucleoli)

  • Search for condition-specific changes in localization or expression

What considerations are important when selecting between different isotypes of FAM222B antibodies for functional studies?

While most commercial FAM222B antibodies are rabbit polyclonal IgG , researchers developing functional antibodies should consider:

• Isotype Selection Based on Function:

  • IgG1 is preferred when Fc-mediated effector functions are desired

  • IgG2 and IgG4 are better when effector functions should be minimized

  • IgG4 with S228P mutation prevents Fab arm exchange with endogenous IgG4

• Fc-Mediated Effector Functions:

  • Antibody-dependent cellular cytotoxicity (ADCC)

  • Antibody-dependent cellular phagocytosis (ADCP)

  • Complement-dependent cytotoxicity (CDC)

• Engineering Considerations:

  • Mutations enhancing FcγRIIIA binding improve ADCC

  • Modifications to FcγRIIA binding affect ADCP

  • Glycosylation patterns (particularly fucosylation) affect effector functions

For most research applications targeting FAM222B, effector functions may not be critical unless studying targeted protein degradation or cellular depletion strategies.

How can I assess and optimize the quality of FAM222B antibodies for reproducible research?

Ensuring antibody quality is essential for reproducible research:

• Characteristic Assessment:

  • Verify antibody specificity through the methods described in 3.1

  • Determine batch-to-batch consistency through standardized assays

  • Assess binding affinity and epitope accessibility in different applications

• Storage and Handling:

  • Follow manufacturer recommendations (typically 4°C short term, -20°C long term)

  • Avoid repeated freeze-thaw cycles

  • Aliquot antibodies to minimize degradation

  • Include preservatives (e.g., 0.02% sodium azide) for long-term storage

• Reporting Standards:

  • Document complete antibody information in publications:

    • Supplier and catalog number

    • Clone/lot number

    • Host species and isotype

    • Validated applications and dilutions

    • Positive and negative controls used

• Validation Best Practices:

  • Use orthogonal methods to confirm specificity

  • Consider genetic knockdown/knockout validation

  • Include proper controls in all experiments

  • Test antibody performance across multiple lots when possible