RCBTB1 Antibody

What is RCBTB1 and why is it important to study?

RCBTB1 (Regulator of Chromosome Condensation and BTB Domain Containing Protein 1) is a 531 amino acid protein that plays crucial roles in cell cycle regulation through chromatin remodeling . The protein contains two key structural elements: two BTB (POZ) domains and six RCC1 repeats, which are essential for its function in transcriptional regulation and chromatin structure control . RCBTB1 is primarily localized in the nucleus and may interact with specific proteins involved in chromatin dynamics, thereby influencing gene expression and cellular proliferation .

The gene is located on human chromosome 13, a region often deleted in B-cell chronic lymphocytic leukemia, suggesting a potential role in tumor suppression . Recent research has identified biallelic mutations in RCBTB1 as causes of retinal dystrophy, sometimes associated with extra-ocular manifestations including goiter, primary ovarian insufficiency, and mild intellectual disability . In the retina specifically, RCBTB1 may act as a substrate adaptor in the ubiquitinylation pathway and possibly modify the localization of oxidative stress-response transcription factors .

What types of RCBTB1 antibodies are available for research?

There are several types of RCBTB1 antibodies available for research purposes, varying in their host organisms, clonality, target epitopes, and applications. These include:

Host organisms:

• Rabbit polyclonal antibodies (most common)

• Mouse monoclonal antibodies (e.g., G-2 clone)

Clonality:

• Polyclonal antibodies recognizing multiple epitopes

• Monoclonal antibodies (e.g., clone 1E4) targeting specific epitopes

Target regions:

• Antibodies targeting various regions of RCBTB1:

  • Internal region

  • N-terminal region (AA 2-90)

  • C-terminal region (AA 350-377)

  • Central region (AA 251-300)

  • Full-length protein (AA 1-355)

Conjugation options:

• Unconjugated primary antibodies

• Conjugated forms including:

  • Horseradish peroxidase (HRP)

  • Phycoerythrin (PE)

  • Fluorescein isothiocyanate (FITC)

  • Various Alexa Fluor® conjugates

  • Agarose for immunoprecipitation

How do I select the appropriate RCBTB1 antibody for my specific research application?

Selecting the appropriate RCBTB1 antibody depends on several factors related to your experimental design and objectives:

1. Intended application:

• For Western blotting: Most RCBTB1 antibodies work well; consider unconjugated or HRP-conjugated options

• For immunohistochemistry: Choose antibodies validated for paraffin-embedded sections

• For immunofluorescence: Select antibodies specifically validated for IF applications

• For immunoprecipitation: Consider agarose-conjugated antibodies

• For ELISA: Ensure the antibody is validated for this application

2. Species reactivity:

• Ensure the antibody reacts with your species of interest (human, mouse, rat, etc.)

• Some antibodies offer broader cross-reactivity (e.g., human, mouse, dog, monkey, pig)

3. Target region relevance:

• Consider which domain of RCBTB1 is most relevant to your research

• For full protein detection, antibodies targeting conserved regions work well

• For studying specific mutations or variants, choose antibodies that don't target the affected region

4. Clonality considerations:

• Polyclonal antibodies: Better for general detection and higher sensitivity

• Monoclonal antibodies: Superior specificity and consistency between batches

5. Validation evidence:

• Review provided application data from manufacturers

• Check for literature citations using the specific antibody

What are the optimal conditions for Western blotting with RCBTB1 antibodies?

Sample preparation:

• Cell/tissue lysis: Use RIPA buffer supplemented with protease inhibitors

• Protein quantification: Bradford or BCA assay to ensure equal loading

• Sample reduction: Add β-mercaptoethanol to sample buffer and heat at 95°C for 5 minutes

Gel electrophoresis parameters:

• Protein amount: 20-40 μg per lane

• Gel percentage: 10% SDS-PAGE (optimal for resolving 531 amino acid RCBTB1 protein)

• Running conditions: 100-120V for approximately 1.5 hours

Transfer conditions:

• Membrane: PVDF (preferred) or nitrocellulose

• Transfer method: Wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Buffer: Standard Towbin buffer with 20% methanol

Antibody incubation:

• Blocking: 5% non-fat dry milk in TBS-T, 1 hour at room temperature

• Primary antibody: Dilute RCBTB1 antibody to manufacturer-recommended concentration (typically 1:500-1:1000)

• Incubation: Overnight at 4°C with gentle rocking

• Secondary antibody: Anti-rabbit or anti-mouse HRP-conjugated (1:5000-1:10000), 1 hour at room temperature

Detection:

• Method: Enhanced chemiluminescence (ECL)

• Expected band size: Approximately 58-60 kDa

• Positive controls: Lysates from cell lines with known RCBTB1 expression

How can I optimize immunohistochemistry protocols for RCBTB1 detection in retinal tissue sections?

Given the importance of RCBTB1 in retinal function and its association with retinal dystrophies , optimizing IHC protocols for retinal tissue is particularly valuable:

Tissue processing:

• Fixation: 4% paraformaldehyde for 24 hours

• Embedding: Paraffin embedding with careful orientation to preserve retinal layers

• Sectioning: 5-7 μm sections on positively charged slides

Antigen retrieval (critical step):

• Method: Heat-induced epitope retrieval

• Buffer options:

  • Citrate buffer (pH 6.0): 10 mM sodium citrate, heat to 95-100°C for 20 minutes

  • EDTA buffer (pH 9.0): May provide better results for some RCBTB1 epitopes

• Cooling: Allow slides to cool gradually at room temperature for 20 minutes

Blocking and antibody incubation:

• Peroxidase block: 3% hydrogen peroxide, 10 minutes

• Protein block: 5-10% normal serum (species of secondary antibody), 1 hour

• Primary antibody: RCBTB1 antibody diluted 1:100-1:200

• Incubation: Overnight at 4°C in humidified chamber

• Secondary antibody: HRP-conjugated, 30-60 minutes at room temperature

• Detection: DAB substrate, monitor under microscope for optimal signal

Counterstaining and controls:

• Hematoxylin for nuclear counterstain, 30-60 seconds

• Positive control: Normal retinal tissue with known RCBTB1 expression

• Negative control: Omit primary antibody

• Comparative analysis: Include diseased tissue with RCBTB1 mutations when available

What approaches should be used for quantitative analysis of RCBTB1 expression across different cellular compartments?

Subcellular fractionation followed by Western blotting:

• Perform subcellular fractionation to isolate nuclear, cytoplasmic, and membrane fractions

• Confirm fractionation quality using compartment-specific markers (e.g., Lamin A/C for nucleus)

• Run equal protein amounts from each fraction on SDS-PAGE

• Probe with RCBTB1 antibody and quantify relative expression

Quantitative immunofluorescence approaches:

• Perform immunofluorescence staining with RCBTB1 antibody

• Counterstain with compartment-specific markers:

  • DAPI for nucleus

  • Specific organelle markers as needed

• Acquire high-resolution confocal z-stack images

• Use image analysis software to:

  • Define subcellular regions based on marker staining

  • Measure RCBTB1 fluorescence intensity in each compartment

  • Calculate ratios between compartments

Flow cytometry for RCBTB1 quantification:

• Fix and permeabilize cells appropriately (based on planned detection of nuclear RCBTB1)

• Stain with RCBTB1 antibody conjugated to fluorophore or use secondary antibody approach

• Include proper controls:

  • Isotype control

  • Secondary-only control

  • Positive and negative cell lines

• Analyze data to determine RCBTB1 expression levels across your samples

How can I address issues with non-specific binding when using RCBTB1 antibodies?

Common non-specific binding issues:

• Multiple bands in Western blot

• High background in immunofluorescence

• Non-specific staining in IHC/ICC

Optimization strategies:

Validation approaches:

• Perform knockout/knockdown controls to confirm specificity

• Use multiple antibodies targeting different RCBTB1 epitopes

• Include competing peptide controls for peptide-derived antibodies

What are the best approaches for dual/multiple labeling with RCBTB1 antibodies?

Planning considerations:

• Antibody host species compatibility

• Fluorophore selection to avoid spectral overlap

• Nuclear localization of RCBTB1 and potential co-localization targets

Recommended protocols:

For immunofluorescence:

• Sequential staining approach:

  • First primary antibody (e.g., RCBTB1) overnight at 4°C

  • First secondary antibody (1 hour, room temperature)

  • Blocking step with normal serum of first primary host

  • Second primary antibody

  • Second secondary antibody

• Host-based simultaneous approach (if primaries are from different hosts):

  • Incubate with both primary antibodies simultaneously

  • Wash thoroughly

  • Incubate with spectrally distinct secondary antibodies

Controls required:

• Single-stained controls for each antibody

• Secondary-only controls

• Fluorophore compensation controls if using confocal microscopy

Recommended co-staining targets for RCBTB1:

• Nuclear markers to confirm localization (e.g., DAPI)

• Cell cycle markers (given RCBTB1's role in cell cycle regulation)

• Ubiquitination pathway components (based on putative function in retina)

• Oxidative stress response factors

How can I validate RCBTB1 antibody specificity for my specific experimental system?

Genetic validation approaches:

• CRISPR/Cas9 knockout of RCBTB1

  • Compare antibody signal in wild-type vs. knockout cells

  • Complete loss of signal indicates high specificity

• siRNA/shRNA knockdown

  • Quantify reduction in signal corresponding to knockdown efficiency

  • Western blot should show proportional reduction in band intensity

Biochemical validation:

• Peptide competition assay

  • Pre-incubate antibody with immunizing peptide

  • Signal should be significantly reduced or eliminated

• Immunoprecipitation followed by mass spectrometry

  • IP using RCBTB1 antibody

  • Confirm RCBTB1 as major identified protein by MS

Cross-antibody validation:

• Compare results using multiple antibodies targeting different RCBTB1 epitopes

  • Similar patterns increase confidence in specificity

  • Different results suggest potential isoform detection or non-specific binding

• Recombinant expression system

  • Overexpress tagged RCBTB1 in cells

  • Detect with both tag antibody and RCBTB1 antibody

  • Co-localization confirms specificity

How do RCBTB1 mutations affect protein detection by different antibodies?

The impact of RCBTB1 mutations on antibody detection depends on the mutation type and location relative to the antibody's target epitope. Based on the reported RCBTB1 variants associated with retinal dystrophy , researchers should consider:

Epitope accessibility considerations:

• Missense mutations (e.g., p.Ser342Leu, p.Pro224Leu) may alter protein conformation

• Protein conformational changes could mask epitopes even distant from mutation site

• Premature stop codons (e.g., p.Gln120*) generate truncated proteins

Antibody selection strategies for mutation studies:

Validation approaches for mutant proteins:

• Express wild-type and mutant constructs in cell models

• Test detection with different RCBTB1 antibodies

• Compare expression patterns and levels

• Consider using epitope-tagged constructs as controls

What are the best experimental designs for studying RCBTB1's role in retinal disease models?

Given the established role of RCBTB1 mutations in retinal dystrophies , several experimental approaches are valuable:

Cellular models:

• Patient-derived iPSCs differentiated to retinal organoids

  • Compare RCBTB1 localization and expression between patient and control

  • Assess impact on retinal cell development and survival

  • Monitor ubiquitination pathway and oxidative stress responses

• CRISPR/Cas9 knock-in of specific mutations in retinal cell lines

  • Create isogenic lines with disease-causing mutations

  • Study impact on RCBTB1 function and downstream pathways

  • Evaluate cell cycle regulation and chromatin structure

Animal models:

• RCBTB1 knockout or knock-in mice

  • Characterize retinal phenotype using ERG, OCT, and histology

  • Assess progression of retinal degeneration

  • Investigate extra-ocular manifestations reported in patients

• Conditional knockout approaches

  • Retina-specific RCBTB1 deletion to isolate retinal phenotypes

  • Temporal control to study developmental vs. degenerative roles

Molecular interaction studies:

• IP-MS to identify RCBTB1 interactors in retinal tissue

  • Compare wild-type vs. mutant interaction networks

  • Focus on ubiquitination and oxidative stress pathways

• Chromatin immunoprecipitation (ChIP) studies

  • Identify genomic regions associated with RCBTB1

  • Assess impact of mutations on chromatin binding

How can RCBTB1 antibodies be used to distinguish between wild-type and mutant protein function in cellular assays?

Functional localization studies:

• Combined immunofluorescence approaches:

  • RCBTB1 antibody staining in patient-derived vs. control cells

  • Co-localization with functional partners

  • Quantitative analysis of nuclear vs. cytoplasmic distribution

• Live-cell imaging approaches:

  • GFP-tagged wild-type vs. mutant RCBTB1

  • Monitor dynamics during cell cycle progression

  • FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

Protein-protein interaction analyses:

• Proximity ligation assay (PLA):

  • Detect endogenous interactions between RCBTB1 and partners

  • Compare interaction patterns between wild-type and mutant

  • Quantify differences in interaction strength

• Co-immunoprecipitation with RCBTB1 antibodies:

  • Pull down RCBTB1 protein complexes from wild-type vs. mutant cells

  • Western blot for known or suspected interaction partners

  • Mass spectrometry to identify differential interactions

Functional assays:

• Ubiquitination pathway function:

  • Assess substrate ubiquitination in wild-type vs. mutant backgrounds

  • Measure proteasomal degradation of target proteins

  • Use RCBTB1 antibodies to monitor RCBTB1 levels and localization

• Oxidative stress response:

  • Challenge cells with oxidative stressors

  • Monitor RCBTB1 localization changes using antibodies

  • Measure downstream transcriptional responses