HEC1/HEC Antibody (Rabbit mAb) [H9F1]

Application: Reactivity: Human

Usage Information

Dilution
WB1:500-1:1000

Application
WB

Reactivity
Human

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
74 kDa

Positive Control	Human spleen tissue; Jurkat cells; HeLa cells; NCCIT cells
Negative Control

Datasheet & SDS

Biological Description

Specificity
HEC1/HEC Antibody (Rabbit mAb) [H9F1] detects endogenous levels of total HEC1/HEC protein.

Clone
H9F1

Synonym(s)
HEC, HEC1, KNTC2, NDC80, Kinetochore protein NDC80 homolog, Highly expressed in cancer protein, Kinetochore protein Hec1, Kinetochore-associated protein 2, Retinoblastoma-associated protein HEC, HsHec1

Background
HEC1, also designated NDC80, is a core structural subunit of the four-protein NDC80 complex, which additionally comprises NUF2, SPC24, and SPC25, and this complex forms the essential, evolutionarily conserved point of physical contact between kinetochores and spindle microtubules during mitosis. The complex assembles into a highly elongated, rod-shaped structure roughly sixty nanometers in length, built from paired coiled-coil regions with globular functional domains positioned at either end; the N-terminal globular domains of HEC1 and NUF2 together fold into a paired calponin-homology domain, a fold structurally related to the microtubule-binding domain of the plus-end-tracking protein EB1, while HEC1 additionally carries an unstructured, positively charged N-terminal tail extending beyond this domain. Microtubule engagement occurs through this calponin-homology interface, which binds cooperatively and predominantly through electrostatic contacts between positive charges on the CH domains and the HEC1 tail and negative charges on the acidic C-terminal tails of tubulin, and gene-silencing and rescue experiments in human cells show that the HEC1 CH domain, the NUF2 CH domain, and the HEC1 tail each contribute to kinetochore-microtubule attachment through mechanistically distinct roles rather than functioning redundantly. The unstructured HEC1 tail specifically increases the binding affinity of purified N-terminal HEC1 fragments and reconstituted NDC80 complexes for microtubules in vitro, and its removal significantly reduces this affinity, while point mutations introduced into the CH domain itself prevent kinetochores from achieving productive microtubule attachment and chromosome alignment in cells, and this CH-domain-mutant phenotype cannot be rescued by blocking Aurora B-mediated phosphorylation of the tail, indicating that the CH domain and the tail perform separable functions that cannot substitute for one another. This attachment is directly regulated by Aurora B kinase, which phosphorylates multiple sites within the disordered HEC1 tail domain and thereby decreases the NDC80 complex's binding affinity for microtubules, weakening kinetochore-microtubule attachments specifically at kinetochores lacking proper tension or bipolar orientation, providing the core error-correction mechanism that allows incorrect attachments to detach and be remade. This tension-sensitive, Aurora B-tunable binding affinity operates upstream of the spindle assembly checkpoint, since NUF2 and HEC1 are additionally required for retention of the checkpoint proteins MAD1 and MAD2 at kinetochores, coupling the physical strength of microtubule attachment directly to the surveillance mechanism that delays anaphase onset until all chromosomes achieve stable, bioriented attachment, disruption of which produces the chromosomal instability and aneuploidy characteristic of many cancers.

Background

HEC1, also designated NDC80, is a core structural subunit of the four-protein NDC80 complex, which additionally comprises NUF2, SPC24, and SPC25, and this complex forms the essential, evolutionarily conserved point of physical contact between kinetochores and spindle microtubules during mitosis. The complex assembles into a highly elongated, rod-shaped structure roughly sixty nanometers in length, built from paired coiled-coil regions with globular functional domains positioned at either end; the N-terminal globular domains of HEC1 and NUF2 together fold into a paired calponin-homology domain, a fold structurally related to the microtubule-binding domain of the plus-end-tracking protein EB1, while HEC1 additionally carries an unstructured, positively charged N-terminal tail extending beyond this domain. Microtubule engagement occurs through this calponin-homology interface, which binds cooperatively and predominantly through electrostatic contacts between positive charges on the CH domains and the HEC1 tail and negative charges on the acidic C-terminal tails of tubulin, and gene-silencing and rescue experiments in human cells show that the HEC1 CH domain, the NUF2 CH domain, and the HEC1 tail each contribute to kinetochore-microtubule attachment through mechanistically distinct roles rather than functioning redundantly. The unstructured HEC1 tail specifically increases the binding affinity of purified N-terminal HEC1 fragments and reconstituted NDC80 complexes for microtubules in vitro, and its removal significantly reduces this affinity, while point mutations introduced into the CH domain itself prevent kinetochores from achieving productive microtubule attachment and chromosome alignment in cells, and this CH-domain-mutant phenotype cannot be rescued by blocking Aurora B-mediated phosphorylation of the tail, indicating that the CH domain and the tail perform separable functions that cannot substitute for one another. This attachment is directly regulated by Aurora B kinase, which phosphorylates multiple sites within the disordered HEC1 tail domain and thereby decreases the NDC80 complex's binding affinity for microtubules, weakening kinetochore-microtubule attachments specifically at kinetochores lacking proper tension or bipolar orientation, providing the core error-correction mechanism that allows incorrect attachments to detach and be remade. This tension-sensitive, Aurora B-tunable binding affinity operates upstream of the spindle assembly checkpoint, since NUF2 and HEC1 are additionally required for retention of the checkpoint proteins MAD1 and MAD2 at kinetochores, coupling the physical strength of microtubule attachment directly to the surveillance mechanism that delays anaphase onset until all chromosomes achieve stable, bioriented attachment, disruption of which produces the chromosomal instability and aneuploidy characteristic of many cancers.

References
https://pubmed.ncbi.nlm.nih.gov/18455984/ https://pubmed.ncbi.nlm.nih.gov/21270439/

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%