CCDC28B Antibody

Introduction to CCDC28B Antibody

CCDC28B antibodies are immunological reagents specifically designed to recognize and bind to the CCDC28B protein (Coiled-coil domain-containing protein 28B). The target of these antibodies, CCDC28B, was originally identified as a second site modifier in the ciliopathy Bardet-Biedl syndrome (BBS) . CCDC28B is a protein that localizes to centrosomes and basal bodies and interacts with several BBS-associated proteins . It plays important roles in regulating cilia length through interactions with specific cellular components including MAPKAP1/SIN1 and the kinesin 1 molecular motor . Additionally, recent research has revealed CCDC28B's involvement in immune synapse assembly in non-ciliated T-cells, highlighting its diverse cellular functions .

CCDC28B antibodies serve as crucial tools for studying this protein's expression, localization, and interactions in various cellular contexts, thereby advancing our understanding of its biological functions and potential implications in disease states.

Structure and Properties of CCDC28B Protein

Understanding the structure and properties of the CCDC28B protein is essential for appreciating the characteristics of antibodies directed against it.

Molecular Structure

CCDC28B is a relatively small protein comprised of approximately 200-241 amino acids depending on the isoform . The gene encoding CCDC28B in humans (CCDC28B, Gene ID: 79140) is located on chromosome 1 and consists of six exons . In mice, the Ccdc28b gene (Gene ID: 66264) is located on chromosome 4 . The protein contains a characteristic coiled-coil domain, which facilitates protein-protein interactions.

Cellular Localization and Function

CCDC28B primarily localizes to centrosomes and basal bodies, consistent with its role in ciliogenesis . Additionally, it has been detected in association with early endosomes (Rab5+) in T-cells, where it plays a role in immune synapse assembly . Functionally, CCDC28B regulates cilia length through interactions with multiple proteins, including SIN1 (part of the mTORC2 complex) and kinesin 1 components (KIF5B and KLC1) .

Production Methods for CCDC28B Antibody

CCDC28B antibodies can be produced using various methods, each with distinct advantages and limitations. These production techniques can significantly influence the specificity, sensitivity, and applications of the resulting antibodies.

Hybridoma Technology

The traditional approach for monoclonal antibody production is hybridoma technology, first developed by Köhler and Milstein in 1975 . This method involves:

1. Immunizing mice or other animals with CCDC28B protein or peptides

2. Isolating B-lymphocytes from the spleen of immunized animals

3. Fusing these B-lymphocytes with myeloma cells lacking the HGPRT gene

4. Selecting hybridoma cells in hypoxanthine-aminopterin-thymidine medium

5. Screening for clones producing antibodies with high specificity for CCDC28B

6. Expanding selected clones for antibody production

While this technology produces highly specific monoclonal antibodies, it has limitations including being time-consuming, expensive, and carrying risks of contamination and low cell viability .

Recombinant Antibody Production

Recombinant DNA technology offers an alternative approach for CCDC28B antibody production:

1. Cloning antibody-coding genes into mammalian expression vectors

2. Introducing these vectors into expression hosts such as HEK 293 or CHO-K1 cells

3. Harvesting and purifying the expressed antibodies

This method preserves correct post-translational modifications and can be scaled for larger production volumes .

Novel Production Methods

Several newer techniques have emerged for antibody production:

1. Phage Display Technology: Creates libraries of antibody fragments displayed on bacteriophage surfaces, allowing for selection of high-affinity binders to CCDC28B

2. Single B Cell Technology: Isolates individual B cells from peripheral blood or lymphoid tissues, clones Ig heavy and light chains using RT-PCR, and expresses them in mammalian cells

3. E. coli-Based Production: A simple method for producing functional antibody fragments using bacterial expression systems, as described in recent research

4. Cell-Free Protein Synthesis: Allows rapid production and screening of antibody candidates without involving living cells, eliminating the need for transfection and cloning steps

Each of these methods offers distinct advantages in terms of speed, cost, and scalability for producing CCDC28B antibodies.

Proteintech CCDC28B Antibody (11530-1-AP)

Table 1: Specifications for Proteintech CCDC28B Antibody (11530-1-AP)

Novus Biologicals CCDC28B Antibody (NBP2-15747)

Table 2: Specifications for Novus Biologicals CCDC28B Antibody (NBP2-15747)

Validated Applications and Performance

Published research and manufacturer validation studies have demonstrated successful use of CCDC28B antibodies in multiple applications:

1. Western Blot: Detection of CCDC28B protein in cell and tissue lysates, with bands typically observed at 30-35 kDa

2. Immunocytochemistry/Immunofluorescence: Localization of CCDC28B to centrosomes and endosomes in various cell types

3. Immunohistochemistry: Detection of CCDC28B expression in tissue sections, including validated performance in human gastric cancer samples

4. Immunoprecipitation: Isolation of CCDC28B and its interacting partners, as demonstrated in studies investigating kinesin 1 interactions

Applications of CCDC28B Antibody in Research

CCDC28B antibodies have been instrumental in advancing our understanding of this protein's functions through various research applications. This section highlights key experimental approaches where these antibodies have proven valuable.

Protein Expression Analysis

Western blot analysis using CCDC28B antibodies has been critical for:

1. Confirming CCDC28B expression in different tissues and cell types

2. Validating knockout or knockdown models, such as the Ccdc28b mutant mouse model

3. Detecting variations in protein levels across different experimental conditions

For example, in the generation of a Ccdc28b mutant mouse line using CRISPR/Cas9, western blot analysis with CCDC28B antibodies confirmed the absence of the expected ~22 kDa CCDC28B band in mutant samples, validating the success of the genetic modification .

Protein Localization Studies

Immunocytochemistry and immunofluorescence techniques using CCDC28B antibodies have revealed:

1. The centrosomal localization of CCDC28B in ciliated cells

2. CCDC28B's presence at early endosomes in T-cells

3. Dynamic changes in CCDC28B distribution in response to cellular stimuli

These localization studies have been crucial in connecting CCDC28B's subcellular distribution to its functional roles in ciliogenesis and immune synapse assembly.

Protein Interaction Studies

Immunoprecipitation with CCDC28B antibodies has uncovered important protein-protein interactions:

1. The interaction between CCDC28B and SIN1, a component of the mTORC2 complex

2. CCDC28B's association with kinesin light chain 1 (KLC1) and the heavy chain KIF5B

3. Connections between CCDC28B and retromer-associated proteins like FAM21 and WASH

For instance, researchers used anti-CCDC28B VHH antibodies to immunoprecipitate endogenous CCDC28B and confirmed its interaction with KIF5B, KLC1, and α-tubulin through western blot analysis .

Functional Studies

CCDC28B antibodies have been essential tools in functional studies investigating:

1. The role of CCDC28B in cilia length regulation

2. CCDC28B's function in T-cell immune synapse assembly

3. The consequences of CCDC28B depletion or mutation on cellular processes

These studies have significantly advanced our understanding of CCDC28B's diverse roles in cellular physiology and potential contributions to disease states.

Research Findings Using CCDC28B Antibody

CCDC28B antibodies have enabled several significant discoveries about this protein's functions and disease associations. This section highlights key research findings facilitated by CCDC28B antibodies.

CCDC28B in Cilia Length Regulation

Research using CCDC28B antibodies has revealed that:

1. CCDC28B depletion leads to shortened cilia in various cell types

2. CCDC28B interacts with SIN1 to regulate cilia length through a mechanism independent of mTORC2 signaling

3. The kinesin 1 motor complex (KIF5B and KLC1) interacts with CCDC28B and regulates its subcellular distribution

4. Depletion of kinesin 1 components results in abnormally elongated cilia, suggesting a complex regulatory network involving CCDC28B

These findings have established CCDC28B as a key regulator of ciliogenesis, with potential implications for ciliopathies like Bardet-Biedl syndrome.

CCDC28B in Bardet-Biedl Syndrome

Studies utilizing CCDC28B antibodies have contributed to our understanding of its role in Bardet-Biedl syndrome:

1. A CCDC28B mutant mouse (Ccdc28b mut) created using CRISPR/Cas9 exhibited mild phenotypes compared to typical BBS models

2. While Ccdc28b mut mice did not develop retinal degeneration or obesity (hallmark features of BBS), they showed clear social interaction defects and stereotypical behaviors

3. The mild phenotype suggests that CCDC28B functions primarily as a modifier rather than a causative gene in BBS

These studies support the hypothesis that CCDC28B mutations may modify the severity of BBS phenotypes when present alongside mutations in primary BBS genes.

CCDC28B in mTORC2 Regulation

Research employing CCDC28B antibodies has uncovered its role in mTORC2 signaling:

1. CCDC28B positively regulates mTORC2 by participating in its assembly and stability

2. Depletion of CCDC28B reduces the interaction between SIN1, Rictor, and mTOR, key components of the mTORC2 complex

3. CCDC28B modulates mTORC2 activity, potentially enhancing AKT1 phosphorylation

4. This function appears distinct from CCDC28B's role in cilia length regulation

These findings suggest that CCDC28B plays a dual role in cellular physiology, influencing both ciliogenesis and mTOR signaling.

CCDC28B in Immune Function

Recent research using CCDC28B antibodies has revealed an unexpected role in immune cells:

1. A CVID-associated variant in CCDC28B affects immune synapse assembly in non-ciliated T-cells

2. CCDC28B participates in immune synapse assembly by regulating polarized T-cell antigen receptor (TCR) recycling

3. CCDC28B promotes actin polymerization at endosomal TCRs by recruiting the actin regulator WASH and its partner FAM21

4. This mechanism is essential for sustaining signaling during T-cell activation

These discoveries expand our understanding of CCDC28B beyond cilia-related functions and suggest potential implications for immune disorders.

Future Perspectives in CCDC28B Antibody Research

The field of CCDC28B antibody research continues to evolve, with several promising directions for future investigation:

Advanced Antibody Technologies

Emerging antibody technologies may enhance CCDC28B research:

1. Development of recombinant monoclonal antibodies from E. coli could provide simpler, more rapid production systems for CCDC28B-specific antibodies

2. Cell-free protein synthesis methods may enable faster screening of multiple antibody candidates against different CCDC28B epitopes

3. Camelid single-domain antibodies (nanobodies) may offer improved access to conformational epitopes and enhanced performance in certain applications

Expanding Clinical Applications

CCDC28B antibodies may find expanded applications in clinical research:

1. Investigation of CCDC28B as a potential biomarker in ciliopathies and related disorders

2. Exploration of CCDC28B's role in immune disorders beyond CVID

3. Development of diagnostic applications based on CCDC28B expression patterns or modifications

Novel Research Directions

Several promising research directions may benefit from CCDC28B antibodies:

1. Further characterization of CCDC28B's dual roles in ciliogenesis and immune function

2. Investigation of tissue-specific functions and expression patterns

3. Exploration of potential therapeutic approaches targeting CCDC28B in relevant disease states