CABP5 Antibody

What is CABP5 and where is it primarily expressed?

CABP5 is a neuronal calmodulin-like calcium-binding protein expressed predominantly in the retina and cochlea. In the retina, CABP5 is found in rod bipolar cells and both ON and OFF cone bipolar cells in human and monkey retina. CABP5 belongs to the calcium-binding protein family and plays roles in calcium signaling pathways critical for neuronal function, particularly in sensory systems .

What are the known physiological interactions of CABP5?

Research has identified several important physiological interacting partners for CABP5. Studies using affinity chromatography followed by mass spectrometry and yeast two-hybrid screening have shown that CABP5 interacts with Munc18-1 and myosin VI, two proteins involved in the synaptic vesicle cycle. CABP5 also directly interacts with the calmodulin-binding domain of Cav1.2 L-type calcium channels, which are expressed in rod bipolar cells . These interactions suggest CABP5 plays an important role in vesicle exocytosis and calcium channel regulation.

What methods can be used to identify CABP5-interacting proteins?

Multiple complementary approaches have been successfully employed to identify CABP5-interacting proteins:

• Affinity Chromatography with Mass Spectrometry: Immobilize purified CABP5 on a column, pass retinal extract through it, elute bound proteins, separate by SDS-PAGE, and identify by LC-MS/MS after trypsin digestion. This approach can be performed in both Ca²⁺-containing and Ca²⁺-free conditions to determine calcium-dependency of interactions .

• Yeast Two-Hybrid Screening: Screen a retinal cDNA library using full-length CABP5 as bait. This method identified Munc18-1 as a physiologically relevant interaction partner. When using this approach, multiple reporter genes and stringent selection conditions (e.g., media depleted of leucine, tryptophan, histidine, and adenine with X-gal) should be employed to minimize false positives .

• Coimmunoprecipitation: Precipitate protein complexes from retinal extracts using antibodies against CABP5 or potential binding partners, then detect the interacting proteins by Western blotting. This approach confirms interactions at physiological concentrations and can assess calcium-dependency of interactions .

What are the optimal conditions for using CABP5 antibodies in Western blotting?

For Western blot applications with CABP5 antibodies, the following conditions are recommended:

• Antibody Dilution: 1:500-1:1000 for most commercially available CABP5 antibodies

• Target Molecular Weight: Approximately 19-20 kDa (calculated molecular weight: 19,826 Da)

• Sample Preparation: Tissue homogenates or cell lysates should be prepared in the presence of protease inhibitors to prevent degradation

• Controls: Include positive control tissues (retina extracts) and negative controls (tissues not expressing CABP5)

• Blocking: Standard blocking with 5% non-fat milk or BSA is generally effective

• Storage: Store antibodies at -20°C for long-term storage, and at 4°C for up to one month for frequent use

How can CABP5 antibodies be utilized to study calcium-dependent protein interactions?

When investigating calcium-dependent protein interactions of CABP5:

• Parallel Conditions Testing: Perform affinity chromatography or coimmunoprecipitation experiments both in the presence and absence of Ca²⁺ (typically 1mM CaCl₂ for Ca²⁺-containing conditions) .

• Calcium Chelation: Include EGTA or BAPTA in Ca²⁺-free conditions to ensure complete calcium removal.

• Western Blot Analysis: After separation of proteins by SDS-PAGE, use CABP5 antibodies (1:500-1:1000 dilution) for detection of calcium-dependent interactions .

• Quantification: Compare band intensities between Ca²⁺-containing and Ca²⁺-free conditions to assess the degree of calcium dependency.

Research findings indicate that CABP5 interactions with Munc18-1 and myosin VI appear to be calcium-independent, as these interactions were observed in both Ca²⁺-containing and Ca²⁺-free conditions .

What approaches can be used to study CABP5's role in vesicle exocytosis?

To investigate CABP5's role in vesicle exocytosis:

• Cell Culture Models: Express CABP5 in model systems like NGF-stimulated PC12 cells, which have been shown to exhibit increased neurite outgrowth and dopamine exocytosis when expressing CABP5 .

• Neurotransmitter Release Assays: Measure dopamine release using ELISA in CABP5-expressing versus control cells.

• Immunofluorescence: Use CABP5 antibodies together with markers of synaptic vesicles to visualize colocalization in retinal sections.

• Colocalization Studies: Perform double immunostaining with CABP5 antibodies and antibodies against proteins involved in vesicle exocytosis (e.g., Munc18-1, myosin VI) to map their distribution in bipolar cells .

• Functional Studies: Compare exocytosis in wild-type versus Cabp5-/- knockout mouse retinal cells to assess functional effects .

How can researchers investigate CABP5's modulation of calcium channels?

To study CABP5's effects on calcium channels:

• Electrophysiology: Use patch-clamp recordings in heterologous expression systems (e.g., HEK293T cells) co-expressing CABP5 and calcium channels (Cav1.2 or Cav1.3) to assess:

  • Calcium-dependent inactivation

  • Voltage-dependence of activation

  • Channel kinetics

• Calcium Imaging: Monitor intracellular calcium dynamics in cells expressing CABP5 versus controls.

• Protein Interaction Studies: Use coimmunoprecipitation with CABP5 antibodies to pull down calcium channel subunits from retinal extracts.

• Localization Studies: Perform immunohistochemistry with CABP5 antibodies and calcium channel antibodies to examine their colocalization in retinal bipolar cells .

Research has shown that CABP5 suppresses calcium-dependent inactivation of Cav1.2 channels and shifts the voltage-dependence of activation to more depolarized membrane potentials in transfected HEK293T cells .

How can researchers validate the specificity of CABP5 antibodies?

To ensure CABP5 antibody specificity:

• Knockout/Knockdown Controls: Test antibodies on tissues from Cabp5-/- knockout mice or cells with CABP5 knockdown.

• Peptide Competition Assays: Pre-incubate antibodies with the immunizing peptide before immunostaining or Western blotting; specific signals should be blocked.

• Multiple Antibody Validation: Compare results using different antibodies targeting various epitopes of CABP5.

• Western Blot Verification: Confirm the antibody detects a band of the expected molecular weight (~20 kDa).

• Positive and Negative Control Tissues: Compare staining between tissues known to express CABP5 (retina) and those that do not.

• Recombinant Protein Controls: Test antibodies against purified recombinant CABP5 protein .

What controls should be included in coimmunoprecipitation studies with CABP5 antibodies?

For reliable coimmunoprecipitation experiments:

• Negative Controls:

  • Use preimmune serum or isotype-matched control IgG instead of the specific antibody

  • Use protein G beads alone without antibody

  • Include samples from Cabp5-/- knockout mice

• Reciprocal Coimmunoprecipitation: If CABP5 interacts with protein X, then immunoprecipitation with anti-X antibodies should co-precipitate CABP5, and vice versa (though this is not always possible, as observed with Munc18-1 where anti-CABP5 antibodies may mask the interaction domain) .

• Input Samples: Always include an input sample (pre-immunoprecipitation lysate) on your blots.

• Calcium Conditions: Perform parallel experiments in the presence and absence of calcium to assess calcium dependency.

• Antibody Validation: Ensure your CABP5 antibody is specific using the validation strategies described above .

What is the expression pattern of CABP5 in the retina and its functional significance?

CABP5 shows a specific expression pattern in the retina:

• In human and monkey retina, CABP5 is expressed in rod bipolar cells and both ON and OFF cone bipolar cells .

• CABP5 is also expressed in cochlear inner hair cells .

• Functionally, CABP5 is required for normal transmission of light signals throughout the retina, as evidenced by the reduced sensitivity (by approximately 50%) of retinal ganglion cell light responses in Cabp5-/- knockout mice .

• Despite this functional deficit, no morphological changes or significant differences in ERG amplitudes were observed in knockout mice, suggesting CABP5's role is in modulating sensitivity rather than in gross retinal development or function .

How do CABP5's interactions with synaptic proteins inform its function?

CABP5's interactions with synaptic proteins provide significant insights into its function:

• Interaction with Munc18-1: Munc18-1 is essential for synaptic vesicle docking and fusion. CABP5's interaction with Munc18-1 suggests it may regulate neurotransmitter release from bipolar cells .

• Interaction with Myosin VI: This motor protein is involved in vesicle trafficking. CABP5's interaction with myosin VI points to a role in synaptic vesicle transport or localization .

• Modulation of Calcium Channels: CABP5 suppresses calcium-dependent inactivation of L-type calcium channels (Cav1.2 and Cav1.3), which are important for sustained neurotransmitter release. This suggests CABP5 may enhance sustained synaptic transmission by preventing channel inactivation .

• Effects on PC12 Cells: Expression of CABP5 in NGF-stimulated PC12 cells stimulates neurite outgrowth and dopamine exocytosis, providing further evidence for its role in vesicle exocytosis .

Combined, these interactions suggest CABP5 plays a multifaceted role in regulating synaptic transmission, particularly in retinal bipolar cells, by coordinating calcium signaling with vesicle exocytosis.