HEC1/HEC Antibody (Rabbit mAb) [H9F1] Datasheet

Biological Description

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

Specificity

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

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.

Usage Information

Application

WB

Dilution

WB

1:500-1:1000

Reactivity

Human

Source

Rabbit Monoclonal Antibody

MW

74 kDa

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

Experimental Methods
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Methods

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

References

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

Application Data

WB

Validated by Selleck

Lane 1: Jurka, Lane 2: HeLa, Lane 3: NCCIT