USHBP1 Antibody

What is USHBP1 and why is it important in research?

USHBP1 (Usher Syndrome 1C Binding Protein 1) is a 703 amino acid protein belonging to the MCC family, also known as MCC2 (Mutated in colon cancer protein 2) or AIE-75-binding protein . The protein-coding gene is located on chromosome 19 . USHBP1 shows highest expression in heart tissue, with moderate to low expression in skeletal muscle, kidney, liver, small intestine, placenta, and lung .

The protein is significant in research due to its associations with several diseases, including:

• Colorectal cancer

• Autosomal recessive spondylocostal dysostosis

• Posterior fusion of lumbosacral vertebrae-blepharoptosis syndrome

• Malignant epithelial tumor of ovary

• Klippel-Feil syndrome 3

• Multiple synostoses syndrome 2

• Prata-Liberal-Goncalves syndrome

• Microcephaly-cervical spine fusion anomalies syndrome

Its name suggests a functional relationship with Usher syndrome proteins, making it relevant to hearing loss and vestibular dysfunction research.

What applications are USHBP1 antibodies commonly used for?

USHBP1 antibodies are utilized in multiple experimental applications:

The observed molecular weight in Western blot analysis is typically 90-100 kDa, which differs from the calculated molecular weight of 76 kDa , suggesting possible post-translational modifications.

What types of USHBP1 antibodies are commercially available?

Several types of USHBP1 antibodies are available for research purposes:

Most commercially available USHBP1 antibodies are rabbit polyclonal antibodies that recognize human, mouse, and/or rat USHBP1 . Conjugated antibodies offer direct detection without secondary antibodies, simplifying experimental workflows for specific applications.

How should researchers store and handle USHBP1 antibodies?

Proper storage and handling of USHBP1 antibodies is crucial for maintaining their performance:

Storage conditions:

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

• Some products may require -80°C storage

• Avoid repeated freeze-thaw cycles

Buffer composition:

• Typically provided in PBS (pH 7.3-7.4)

• Contains preservatives such as 0.02% sodium azide

• Often includes stabilizers like glycerol (10-50%)

Safety considerations:

• Sodium azide is a hazardous substance that should be handled by trained personnel only

• Follow manufacturer's recommendations for thawing and aliquoting

Stability:

• Most products are stable for one year after shipment when properly stored

• Some manufacturers indicate that aliquoting is unnecessary for -20°C storage

What are the optimal conditions for using USHBP1 antibodies in Western blot analysis?

For optimal Western blot results with USHBP1 antibodies:

Sample preparation:

• Positive controls: Heart tissue (highest expression), A549 cells, COLO 320 cells

• Protein extraction methods should preserve native conformation when possible

• Include protease and phosphatase inhibitors in lysis buffers

Protocol optimization:

• Working dilution: 1:200-1:1000 (requires empirical determination)

• Expected molecular weight: 90-100 kDa (higher than calculated 76 kDa)

• Blocking: 5% non-fat milk or BSA in TBST is typically effective

• Secondary antibody: Anti-rabbit IgG for most commercial USHBP1 antibodies

Troubleshooting considerations:

• Multiple bands may indicate isoforms or degradation products

• High background may require increased antibody dilution or alternative blocking agents

• Weak signals may be improved with enhanced chemiluminescence systems or longer exposure times

Performing a dilution series experiment is recommended to determine the optimal antibody concentration for your specific experimental system.

What antigen retrieval methods are most effective for USHBP1 immunohistochemistry?

Effective antigen retrieval is critical for successful USHBP1 immunohistochemistry:

Recommended methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative approach: Citrate buffer pH 6.0

Protocol considerations:

• Heat-induced epitope retrieval (HIER) is generally more effective than enzymatic methods

• Duration and temperature may need optimization based on fixation method and tissue type

• Complete immersion of tissue sections in retrieval solution is essential

Tissue-specific validation:

• Successfully detected in human heart tissue and small intestine tissue

• For mouse tissues, validated in heart samples

The effectiveness of antigen retrieval can vary depending on tissue fixation parameters, so preliminary optimization comparing different retrieval methods is recommended when working with new tissue types.

How can researchers validate the specificity of USHBP1 antibodies?

Comprehensive validation of USHBP1 antibodies should include:

Essential validation experiments:

• Western blot showing a single band at expected molecular weight (90-100 kDa)

• Immunostaining patterns consistent with known tissue expression (highest in heart)

• Lack of signal in negative controls (secondary antibody only, isotype controls)

• Comparison across multiple detection methods (WB, IHC, IF)

Advanced validation approaches:

• siRNA knockdown resulting in reduced USHBP1 signal

• Peptide competition assays where pre-incubation with immunizing peptide blocks antibody binding

• Comparison of results from multiple antibodies targeting different USHBP1 epitopes

• Correlation with mRNA expression data

Documentation recommendations:

• Maintain detailed records of validation experiments

• Include positive and negative controls in all experiments

• Report antibody catalog numbers, lot numbers, and dilutions in publications

Recent advances in antibody validation emphasize the importance of using orthogonal methods to confirm specificity beyond traditional approaches .

How do biophysical models inform USHBP1 antibody design and specificity?

Recent research has developed biophysically-informed models to improve antibody design and specificity prediction:

Model approaches:

• Integration of large-scale selection experiments with high-throughput sequencing

• Incorporation of biophysical constraints to offer quantitative insights

• Association of distinct binding modes with specific ligands

Applications to antibody design:

• Prediction of binding sites and epitopes on antigens like USHBP1

• Design of antibodies with custom specificity profiles (highly specific or cross-reactive)

• Identification of off-target binding

Experimental validation:

• Phage display selections against various ligand combinations

• Testing of model-predicted variants not present in training sets

• Evaluation of disentangled binding modes associated with specific ligands

Benefits for USHBP1 research:

• Potential for designing antibodies that discriminate between closely related epitopes

• Reduction in experimental costs through computational prediction

• Enhanced specificity for complex experimental applications

These approaches have demonstrated success in "designing antibodies with tailored specificity, with applications to protein engineering extending beyond the design of antibodies" .

How can active learning strategies improve USHBP1 antibody development?

Active learning offers promising strategies for more efficient antibody development:

Current challenges:

• Out-of-distribution prediction when test antibodies and antigens are not represented in training data

• Limited availability of comprehensive binding datasets due to high experimental costs

• Difficulty in predicting many-to-many relationships between antibodies and antigens

Active learning solutions:

• Start with small labeled datasets and iteratively expand them

• Algorithms can reduce the number of required antigen mutant variants by up to 35%

• Learning process acceleration by up to 28 steps compared to random data selection

Implementation strategies:

• Library-on-library approaches for antibody-antigen binding prediction

• Simulation frameworks like Absolut! for evaluating out-of-distribution performance

• Integration of computational predictions with targeted experimental validation

This approach is particularly valuable for USHBP1 antibody development as it can significantly reduce experimental costs while improving specificity and cross-reactivity profiles.

What factors should be considered when selecting USHBP1 antibodies for disease research?

When investigating USHBP1 in disease contexts, several factors deserve special consideration:

Disease relevance:

• USHBP1 is associated with colorectal cancer and several genetic syndromes

• Expression patterns may differ between normal and disease tissues

• Consider epitopes that may be affected by disease-associated mutations

Model system compatibility:

• Species reactivity: Confirm antibody works in your model organism (human, mouse, rat)

• Validated applications: Ensure antibody is validated for your technique (WB, IHC, IF)

• Tissue-specific considerations: Expression varies across tissues with highest in heart

Technical specifications:

Experimental controls:

• Positive controls: Heart tissue (highest expression), A549 cells, COLO 320 cells

• Negative controls: Secondary antibody only, isotype controls

• Validation in disease context: Preliminary testing in relevant pathological tissues

What approaches can address challenges in USHBP1 detection across species?

Researchers working with USHBP1 across different species should consider:

Cross-reactivity considerations:

• Verify antibody reactivity with human, mouse, and/or rat USHBP1

• Check sequence homology between target epitopes across species

• Some antibodies are species-specific while others cross-react

Protocol adjustments:

• Western blot: Slight molecular weight variations may occur between species

• IHC/IF: Optimization of antigen retrieval and blocking conditions may differ

• Fixation sensitivity can vary between human and rodent samples

Species-specific positive controls:

• Human: A549 cells, COLO 320 cells, heart tissue

• Mouse: Heart tissue, validated cell lines

• Rat: Confirmed reactivity with some antibodies

Dilution optimization:

• Working dilutions should be determined empirically for each species

• Typical ranges:

  • WB: 1:200-1:1000

  • IHC: 1:20-1:200

  • IF/ICC: 1:10-1:100

Initial validation experiments comparing antibody performance across species are highly recommended before proceeding with full experimental series.

What are recommended protocols for immunofluorescence with USHBP1 antibodies?

For successful immunofluorescence detection of USHBP1:

Sample preparation:

• Cell lines: A549 cells have been validated for USHBP1 IF/ICC

• Fixation: 4% paraformaldehyde is commonly used; methanol fixation may be tested as an alternative

• Permeabilization: 0.1-0.5% Triton X-100 in PBS for adequate antibody access

Staining protocol:

• Blocking: 1-5% BSA or normal serum from secondary antibody host species

• Primary antibody dilution: 1:10-1:100 (requires optimization)

• Incubation: Typically overnight at 4°C or 1-2 hours at room temperature

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG (for rabbit primary antibodies)

• Counterstaining: DAPI for nuclear visualization

Advanced approaches:

• Co-staining with other relevant proteins to assess co-localization

• Super-resolution microscopy for detailed subcellular localization

• Live-cell imaging with fluorescently tagged USHBP1 to study dynamics

Controls:

• Positive control: Cells known to express USHBP1 (A549 cells)

• Negative controls: Primary antibody omission, non-specific IgG, cells with low/no expression

For direct detection, FITC-conjugated USHBP1 antibodies are available, which eliminate the need for secondary antibodies .

How can researchers quantify USHBP1 expression levels accurately?

Accurate quantification of USHBP1 requires careful methodological considerations:

Western blot quantification:

• Use appropriate loading controls (β-actin, GAPDH)

• Include a standard curve of recombinant USHBP1 protein when possible

• Analyze band intensity with software like ImageJ or specialized western blot analysis programs

• Normalize USHBP1 signal to loading control for relative quantification

Immunohistochemistry quantification:

• Use digital image analysis software for objective assessment

• Quantify parameters such as staining intensity, percentage of positive cells, or H-score

• Include reference standards on each slide for normalization

ELISA approaches:

• Commercial USHBP1 ELISA kits or develop custom sandwich ELISA using available antibodies

• Include standard curves with recombinant USHBP1 protein

• Technical replicates to assess assay variation

RT-qPCR for complementary mRNA quantification:

• Design specific primers for USHBP1 mRNA

• Use reference genes appropriate for the tissue/cell type

• Correlate protein levels with mRNA expression

Mass spectrometry for absolute quantification:

• Targeted approaches using isotopically labeled standards

• Provides the most accurate absolute quantification

• Can distinguish between specific isoforms or post-translational modifications

What strategies can improve antibody specificity for closely related epitopes?

Recent advances in antibody engineering provide strategies to improve specificity:

Computational approaches:

• Biophysics-informed models that associate distinct binding modes with specific ligands

• Machine learning to predict and optimize antibody-antigen binding

• Design of new antibody sequences with predefined binding profiles

Experimental techniques:

• Phage display selections against diverse ligand combinations

• Epitope mapping to identify unique regions for antibody targeting

• Affinity maturation to increase specificity for the target epitope

Hybrid strategies:

• Starting with experimental data to train computational models

• Using models to generate novel antibody variants

• Experimental validation of predictions in iterative cycles

The integration of these approaches has been shown to "disentangle the different contributions to binding to several epitopes from a single experiment" and enable "the challenging problem of designing new, experimentally untried antibody sequences that discriminate closely related ligands" .

How can researchers troubleshoot common issues with USHBP1 antibodies?

When working with USHBP1 antibodies, researchers may encounter several common challenges:

No signal detected:

• Verify protein expression in your sample (use positive controls like heart tissue)

• Optimize antibody concentration (try a dilution series)

• Check detection system functionality with a known working antibody

• Consider alternative antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

Multiple bands in Western blot:

• Compare to expected molecular weight (90-100 kDa)

• Consider potential isoforms or post-translational modifications

• Evaluate protein degradation (add protease inhibitors)

• Test specificity with blocking peptides

High background:

• Increase blocking concentration or time

• Optimize antibody dilution (typically 1:200-1:1000 for WB)

• Try alternative blocking agents (BSA vs. milk)

• Increase washing duration and number of washes

Inconsistent results:

• Standardize protocols thoroughly

• Use the same lot of antibody when possible

• Maintain consistent sample preparation methods

• Include positive and negative controls in each experiment

Detailed troubleshooting table for specific applications: