INCENP Antibody (Mouse mAb) [L10F19]

Application: Reactivity: Human

Lane 1: Hela, Lane 2: 293T, Lane 3: HCT116, Lane 4: U2OS

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IF1:200

Application
WB, IP, IF

Reactivity
Human

Source
Mouse 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
105 kDa

Positive Control	HeLa cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold Tris-Triton 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 Tris-Triton 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 Tris-Triton 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: 5%. 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 1:1000), 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.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold Tris-Triton 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 Tris-Triton 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 Tris-Triton 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: 5%. 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 1:1000), 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
INCENP Antibody (Mouse mAb) [L10F19] detects endogenous levels of total INCENP protein.

Subcellular Location
Centromere, Chromosome, Cytoplasm, Cytoskeleton, Kinetochore, Microtubule, Nucleus

Uniprot ID
Q9NQS7

Clone
L10F19

Synonym(s)
binds and activates aurora-B and -C in vivo and in vitro; chromosomal passenger; chromosomal passenger protein; inner centromere protein antigens 135/155kDa; inner centromere protein INCENP

Background
INCENP (inner centromere protein) is a core scaffold of the chromosomal passenger complex (CPC) that coordinates chromosome segregation, error correction, and cytokinesis by physically coupling Aurora B kinase to centromeric chromatin and spindle microtubules throughout mitosis. The N-terminal region of INCENP forms a parallel three-helix bundle with Survivin and Borealin, creating the CPC localization module that targets the complex first broadly along chromosome arms, then to inner centromeres at metaphase and finally to the spindle midzone and midbody during anaphase and telophase, while the C-terminal IN-box element wraps around the small lobe of Aurora B and provides the primary activation interface for the catalytic subunit. Human Aurora B bound to the INCENP IN-box shows that INCENP allosterically stabilizes an active-like T-loop conformation, opens the catalytic cleft, and positions key residues in the active site, and that subsequent phosphorylation of two serines in the INCENP C-terminus further shifts the complex into a fully active kinase state, establishing a two-step mechanism in which INCENP first primes and then, when phosphorylated, maximally activates Aurora B. INCENP and Aurora B are mutually dependent: INCENP is required to localize Aurora B correctly and to support its histone H3 Ser10 kinase activity, whereas Aurora B is needed for INCENP accumulation at centromeres and transfer to the spindle at anaphase, and depletion of either component leads to severe defects in histone H3 phosphorylation, metaphase chromosome alignment, sister kinetochore disjunction, and completion of cytokinesis, without completely blocking anaphase onset. INCENP’s central intrinsically disordered region acts as a flexible “dog-leash” that links the centromere-bound CPC core to kinetochore and microtubule substrates; phosphorylation within this disordered segment tunes its elongation and cohesiveness, modulating the reach and dynamic sampling of Aurora B across the inner centromere–kinetochore axis and thereby influencing tension sensing and the spatial error-correction gradient that destabilizes incorrect microtubule–kinetochore attachments. Ablation of INCENP in mitotic cells disrupts recruitment of Aurora B to centromeres, impairs spindle checkpoint signaling, allows cells to exit mitosis with misaligned or lagging chromosomes, and causes frequent cytokinesis failure, all of which contribute to chromosomal instability; in meiosis, INCENP and Aurora B are additionally required for centromeric localization of the shugoshin MEI-S332 and maintenance of sister chromatid cohesion, linking CPC activity to specialized meiotic centromere behavior. Overexpression of INCENP in colorectal and other tumor cell lines, where it colocalizes with Aurora B on metaphase chromosomes and midbodies, and co-overexpression of Aurora B and INCENP in model systems can profoundly disrupt chromosome segregation, consistent with a role for altered CPC stoichiometry in tumor-associated aneuploidy and in modulating sensitivity to Aurora B inhibitors. Across these contexts, INCENP functions as a modular CPC organizer and Aurora B activator whose structured N- and C-terminal domains and phosphorylation-regulated disordered linker together define where and how Aurora B acts on chromatin and microtubule substrates.

Background

INCENP (inner centromere protein) is a core scaffold of the chromosomal passenger complex (CPC) that coordinates chromosome segregation, error correction, and cytokinesis by physically coupling Aurora B kinase to centromeric chromatin and spindle microtubules throughout mitosis. The N-terminal region of INCENP forms a parallel three-helix bundle with Survivin and Borealin, creating the CPC localization module that targets the complex first broadly along chromosome arms, then to inner centromeres at metaphase and finally to the spindle midzone and midbody during anaphase and telophase, while the C-terminal IN-box element wraps around the small lobe of Aurora B and provides the primary activation interface for the catalytic subunit. Human Aurora B bound to the INCENP IN-box shows that INCENP allosterically stabilizes an active-like T-loop conformation, opens the catalytic cleft, and positions key residues in the active site, and that subsequent phosphorylation of two serines in the INCENP C-terminus further shifts the complex into a fully active kinase state, establishing a two-step mechanism in which INCENP first primes and then, when phosphorylated, maximally activates Aurora B. INCENP and Aurora B are mutually dependent: INCENP is required to localize Aurora B correctly and to support its histone H3 Ser10 kinase activity, whereas Aurora B is needed for INCENP accumulation at centromeres and transfer to the spindle at anaphase, and depletion of either component leads to severe defects in histone H3 phosphorylation, metaphase chromosome alignment, sister kinetochore disjunction, and completion of cytokinesis, without completely blocking anaphase onset. INCENP’s central intrinsically disordered region acts as a flexible “dog-leash” that links the centromere-bound CPC core to kinetochore and microtubule substrates; phosphorylation within this disordered segment tunes its elongation and cohesiveness, modulating the reach and dynamic sampling of Aurora B across the inner centromere–kinetochore axis and thereby influencing tension sensing and the spatial error-correction gradient that destabilizes incorrect microtubule–kinetochore attachments. Ablation of INCENP in mitotic cells disrupts recruitment of Aurora B to centromeres, impairs spindle checkpoint signaling, allows cells to exit mitosis with misaligned or lagging chromosomes, and causes frequent cytokinesis failure, all of which contribute to chromosomal instability; in meiosis, INCENP and Aurora B are additionally required for centromeric localization of the shugoshin MEI-S332 and maintenance of sister chromatid cohesion, linking CPC activity to specialized meiotic centromere behavior. Overexpression of INCENP in colorectal and other tumor cell lines, where it colocalizes with Aurora B on metaphase chromosomes and midbodies, and co-overexpression of Aurora B and INCENP in model systems can profoundly disrupt chromosome segregation, consistent with a role for altered CPC stoichiometry in tumor-associated aneuploidy and in modulating sensitivity to Aurora B inhibitors. Across these contexts, INCENP functions as a modular CPC organizer and Aurora B activator whose structured N- and C-terminal domains and phosphorylation-regulated disordered linker together define where and how Aurora B acts on chromatin and microtubule substrates.

References
https://pubmed.ncbi.nlm.nih.gov/15866179/ https://pubmed.ncbi.nlm.nih.gov/28314740/

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%