FAM126B Antibody

What is FAM126B and what biological processes is it involved in?

FAM126B (Family with sequence similarity 126, member B) functions primarily in the phosphatidylinositol 4-kinase pathway. FAM126B works in a functionally redundant manner with FAM126A to facilitate the recruitment of PI4KIIIα to the plasma membrane for phosphatidylinositol 4-phosphate (PI4P) synthesis . PI4P is a critical determinant of plasma membrane identity and plays essential roles in multiple cellular processes. FAM126B is part of a complex that includes PI4KIIIα and the adaptor proteins TTC7 and EFR3, which collectively regulate phosphoinositide metabolism at the plasma membrane .

What are the recommended applications for anti-FAM126B antibodies?

The anti-FAM126B antibody has been validated for several research applications:

• Immunoblotting (Western blotting): Recommended concentration range of 0.04-0.4 μg/mL

• Immunohistochemistry: Recommended dilution range of 1:200-1:500

These applications allow researchers to detect and quantify FAM126B protein expression in various experimental contexts, including tissue samples and cell lysates.

What controls should be included when using FAM126B antibodies?

When using FAM126B antibodies, the following controls are recommended:

• Positive control: Cell lines or tissues known to express FAM126B (such as DLD1 or HCT116 colorectal cancer cell lines) should be included

• Negative control: Consider using samples treated with FAM126B-targeting sgRNAs to create a true negative control

• Loading control: Use established housekeeping proteins (e.g., GAPDH, β-actin) for normalization in immunoblotting experiments

• Isotype control: Include an irrelevant antibody of the same isotype and concentration to assess non-specific binding

How should researchers optimize immunoblotting protocols for detecting FAM126B?

For optimal immunoblotting results with anti-FAM126B antibody:

• Sample preparation: Use RIPA or similar lysis buffers with protease inhibitors for effective protein extraction

• Protein loading: Load 20-40 μg of total protein per lane for consistent detection

• Transfer conditions: Use a wet transfer system with methanol-containing buffer for efficient transfer of FAM126B (~58 kDa)

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

• Antibody concentration: Start with 0.2 μg/mL and adjust as needed based on signal-to-noise ratio

• Incubation: Overnight incubation at 4°C often yields optimal results

• Detection system: ECL-based detection systems are generally suitable, though more sensitive systems may be required for low-expressing samples

What are the key considerations for immunohistochemistry applications with FAM126B antibodies?

For successful immunohistochemical detection of FAM126B:

• Fixation: 10% neutral buffered formalin fixation for 24-48 hours is generally suitable

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking: Use 10% normal serum from the same species as the secondary antibody

• Primary antibody: Dilute at 1:200-1:500 and incubate overnight at 4°C

• Detection system: An appropriate HRP/DAB detection system compatible with rabbit primary antibodies

• Counterstaining: Hematoxylin provides good nuclear contrast

• Controls: Include both positive and negative controls in each staining run

How can researchers validate the specificity of FAM126B antibody detection?

Validating antibody specificity is crucial for reliable results. For FAM126B antibodies, consider:

• Genetic knockdown/knockout: Compare staining between wildtype samples and those with FAM126B depleted via CRISPR-Cas9 or RNAi

• Overexpression: Analyze samples with overexpressed FAM126B to confirm increased signal

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (PFDAPDSTQEGQKVLKVEVTPTVPRISRTAITTASIRRHRWRREGAEGVNGGEESVNLNDADEGFSSGASLSSQPIGTKPSSSS) to block specific binding

• Multiple antibodies: Compare results using different antibodies targeting distinct epitopes

• Molecular weight verification: Confirm detection at the expected molecular weight (~58 kDa)

How can FAM126B antibodies be used to investigate the PI4KIIIα complex in normal and disease states?

The PI4KIIIα complex represents an important area for investigation using FAM126B antibodies:

• Co-immunoprecipitation: Use anti-FAM126B antibodies to pull down the entire PI4KIIIα complex, followed by immunoblotting for other components (PI4KIIIα, TTC7, EFR3)

• Proximity ligation assay: Detect in situ interactions between FAM126B and other complex components in cells or tissues

• Immunofluorescence co-localization: Examine the subcellular distribution of FAM126B in relation to other complex members

• Comparative analysis: Compare complex formation in normal versus disease tissues (particularly colorectal cancer samples)

• Fractionation studies: Analyze membrane versus cytosolic distribution of FAM126B and complex components

These approaches can reveal alterations in complex formation, stability, or localization that may contribute to disease pathogenesis.

What strategies should be employed to investigate FAM126B as a therapeutic target in colorectal cancer?

Research exploring FAM126B as a therapeutic target should consider:

• Expression profiling: Characterize FAM126A and FAM126B expression across cancer types and normal tissues using immunohistochemistry and western blotting

• Functional validation: Employ genetic depletion approaches (CRISPR-Cas9, shRNA) to confirm FAM126B dependency in FAM126A-low cancer cell lines

• In vivo models: Test FAM126B depletion in xenograft models derived from FAM126A-low versus FAM126A-high cell lines

• Mechanism studies: Investigate how FAM126B depletion leads to cell death in FAM126A-low contexts, focusing on PI4P synthesis disruption

• Biomarker development: Establish FAM126A expression as a biomarker for potential FAM126B-targeted therapies

How can researchers investigate the functional redundancy between FAM126A and FAM126B?

To explore the functional relationship between FAM126A and FAM126B:

• Rescue experiments: Express FAM126A in FAM126A-low/FAM126B-dependent cell lines to test if this rescues FAM126B dependency

• Double depletion studies: Compare the effects of individual versus combined depletion of FAM126A and FAM126B on PI4P levels and cell viability

• Domain analysis: Create chimeric proteins or domain deletion constructs to identify regions mediating functional redundancy

• Quantitative PI4P assays: Measure PI4P levels after FAM126A and/or FAM126B manipulation

• Protein stability analysis: Investigate whether FAM126A and FAM126B stabilize each other or other complex components

What are common technical issues when working with FAM126B antibodies and how can they be addressed?

Common challenges and solutions include:

• High background:

  • Increase blocking time/concentration

  • Reduce primary antibody concentration

  • Include additional washing steps

  • Ensure secondary antibody compatibility

• Weak or no signal:

  • Increase antibody concentration

  • Optimize antigen retrieval (for IHC)

  • Extend primary antibody incubation time

  • Consider protein degradation issues during sample preparation

• Non-specific bands:

  • Increase washing stringency

  • Optimize blocking conditions

  • Verify antibody specificity using knockout controls

  • Consider using a different detection system

• Inconsistent results:

  • Standardize lysate preparation

  • Maintain consistent antibody lots

  • Control for experimental variables (cell density, passage number)

  • Implement rigorous quantification methods

How should researchers interpret FAM126B expression data in the context of FAM126A levels?

When analyzing FAM126B expression:

• Always assess FAM126A expression in parallel to provide context for FAM126B data

• Consider the relative abundance of both proteins, as their functional redundancy means that low FAM126A may increase reliance on FAM126B

• Use appropriate cell line models representing different FAM126A/FAM126B expression patterns (e.g., RKO and SW48 for FAM126A-low; DLD1 and HCT116 for FAM126A-high)

• When possible, quantify absolute protein levels rather than relying solely on relative measures

• Interpret functional studies in light of the compensatory relationship between these proteins

What considerations are important when selecting control cell lines for FAM126B studies?

When selecting appropriate controls:

• Characterize FAM126A and FAM126B expression levels in candidate cell lines

• For colorectal cancer research, consider using:

  • RKO and SW48 (FAM126A-low cell lines)

  • DLD1 and HCT116 (FAM126A-high cell lines)

• Generate isogenic cell line pairs through genetic manipulation:

  • FAM126A knockout in FAM126A-high lines

  • FAM126A overexpression in FAM126A-low lines

• Consider tissue of origin when selecting control lines

• Account for other PI4KIIIα complex components that might influence FAM126B function

How might FAM126B antibodies be used to explore the role of PI4P in membrane biology?

FAM126B antibodies can advance PI4P research through:

• Super-resolution microscopy to visualize FAM126B localization at plasma membrane microdomains

• Time-lapse imaging combined with FAM126B immunostaining to track dynamic changes in PI4P synthesis

• Correlative light and electron microscopy to examine FAM126B localization at ultrastructural level

• Investigating FAM126B redistribution during cellular processes requiring membrane remodeling

• Exploring potential non-canonical functions of FAM126B beyond the PI4KIIIα complex

What approaches can be used to study post-translational modifications of FAM126B?

To investigate FAM126B post-translational modifications:

• Phospho-specific antibodies: Develop antibodies recognizing phosphorylated forms of FAM126B

• Mass spectrometry: Perform immunoprecipitation followed by mass spectrometry to identify modification sites

• Mutagenesis studies: Create point mutations at predicted modification sites to assess functional impacts

• Treatment studies: Examine how various signaling pathway modulators affect FAM126B modification status

• In vitro kinase assays: Identify kinases responsible for FAM126B phosphorylation

How can researchers investigate the structural basis of FAM126B function within the PI4KIIIα complex?

To explore structural aspects of FAM126B function:

• Cross-linking mass spectrometry: Map interaction interfaces between FAM126B and other complex components

• Hydrogen-deuterium exchange: Identify regions of FAM126B that undergo conformational changes upon complex formation

• Cryo-electron microscopy: Visualize the entire PI4KIIIα complex including FAM126B

• Structure-guided mutagenesis: Create point mutations in conserved residues to disrupt specific interactions

• Comparative modeling: Build on the FAM126A-TTC7 co-crystal structure to predict FAM126B structural features