Otoferlin Antibody (Rabbit mAb) [P6C24]

Application: Reactivity: Rat, Mouse

Usage Information

Dilution
WB1:1000 IHC1:500

Application
WB, IHC

Reactivity
Rat, Mouse

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
227 kDa 250 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Mouse cochlea tissue; Mouse brain tissue; Rat cochlea tissue; Rat brain tissue
Negative Control	Rat liver tissue; Mouse liver tissue

Datasheet & SDS

Biological Description

Specificity
Otoferlin Antibody (Rabbit mAb) [P6C24] detects endogenous levels of total Otoferlin protein.

Clone
P6C24

Synonym(s)
Fer1l2, Otof, Otoferlin, Fer-1-like protein 2, Protein pachanga

Background
Otoferlin belongs to the ferlin family of large, multi-C2-domain transmembrane proteins and functions as the principal calcium sensor governing synaptic vesicle exocytosis at the ribbon synapses of cochlear and vestibular sensory hair cells, structurally distinguished from the neuronal calcium sensor synaptotagmin by its far larger size and its six tandem C2 domains, designated C2A through C2F, connected to a single C-terminal transmembrane anchor. Recombinant protein analysis shows that five of these six C2 domains directly bind calcium, with apparent dissociation constants in the low micromolar range that increase in calcium affinity by up to sevenfold when the domains are presented in the context of a membrane surface rather than free in solution, and in a reconstituted membrane fusion assay these same five calcium-sensing C2 domains each independently stimulate membrane fusion in a calcium-dependent manner, directly establishing otoferlin's C2 domains as functional calcium-triggered fusion catalysts rather than passive calcium-binding modules. Calcium regulates otoferlin's interactions with the SNARE fusion machinery in a domain-specific manner: the C2F domain binds the target-membrane SNARE protein SNAP-25 and the phospholipid PI(4,5)P2 maximally at high micromolar calcium concentrations with reduced binding in calcium's absence, while C2F additionally engages otoferlin's other C2 domains specifically in the absence of calcium, consistent with a closed, autoinhibited tertiary conformation at low calcium that opens as calcium concentration rises; C2D, C2E, and C2F engage the SNARE protein syntaxin-1 more strongly above roughly ten micromolar calcium, a concentration range matching the elevated calcium levels reached at the hair cell ribbon synapse during active calcium influx, while weaker, calcium-independent interactions with t-SNAREs are proposed to support baseline vesicle docking at resting calcium levels. Otoferlin coimmunoprecipitates syntaxin-1 and SNAP-25 but not the vesicular SNARE synaptobrevin-1, and the C2D domain additionally binds the Cav1.3 subunit of the presynaptic L-type calcium channel, positioning otoferlin to physically couple calcium channel activity at the active zone to SNARE-mediated vesicle fusion through a single multidomain protein.

Background

Otoferlin belongs to the ferlin family of large, multi-C2-domain transmembrane proteins and functions as the principal calcium sensor governing synaptic vesicle exocytosis at the ribbon synapses of cochlear and vestibular sensory hair cells, structurally distinguished from the neuronal calcium sensor synaptotagmin by its far larger size and its six tandem C2 domains, designated C2A through C2F, connected to a single C-terminal transmembrane anchor. Recombinant protein analysis shows that five of these six C2 domains directly bind calcium, with apparent dissociation constants in the low micromolar range that increase in calcium affinity by up to sevenfold when the domains are presented in the context of a membrane surface rather than free in solution, and in a reconstituted membrane fusion assay these same five calcium-sensing C2 domains each independently stimulate membrane fusion in a calcium-dependent manner, directly establishing otoferlin's C2 domains as functional calcium-triggered fusion catalysts rather than passive calcium-binding modules. Calcium regulates otoferlin's interactions with the SNARE fusion machinery in a domain-specific manner: the C2F domain binds the target-membrane SNARE protein SNAP-25 and the phospholipid PI(4,5)P2 maximally at high micromolar calcium concentrations with reduced binding in calcium's absence, while C2F additionally engages otoferlin's other C2 domains specifically in the absence of calcium, consistent with a closed, autoinhibited tertiary conformation at low calcium that opens as calcium concentration rises; C2D, C2E, and C2F engage the SNARE protein syntaxin-1 more strongly above roughly ten micromolar calcium, a concentration range matching the elevated calcium levels reached at the hair cell ribbon synapse during active calcium influx, while weaker, calcium-independent interactions with t-SNAREs are proposed to support baseline vesicle docking at resting calcium levels. Otoferlin coimmunoprecipitates syntaxin-1 and SNAP-25 but not the vesicular SNARE synaptobrevin-1, and the C2D domain additionally binds the Cav1.3 subunit of the presynaptic L-type calcium channel, positioning otoferlin to physically couple calcium channel activity at the active zone to SNARE-mediated vesicle fusion through a single multidomain protein.

References
https://pubmed.ncbi.nlm.nih.gov/20921140/ https://pubmed.ncbi.nlm.nih.gov/24478316/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%