NEK6 Antibody [P9K17] | Primary Antibodies

Application: Reactivity: Human

Lane 1: HepG2, Lane 2: Hela, Lane 3: 293T, Lane 4: HT1080

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000-1:10000

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 Observed MW
36 kDa 36 kDa,53 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	HeLa cells; HepG2 cells; 293T cells; HT1080 cells; HepG2 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: 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, 60 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: 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, 60 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.

Datasheet & SDS

Biological Description

Specificity
NEK6 Antibody (Rabbit mAb) [P9K17] detects endogenous levels of total NEK6 protein.

Subcellular Location
Cytoplasm, Cytoskeleton, Microtubule

Uniprot ID
Q9HC98

Clone
P9K17

Synonym(s)
Serine/threonine-protein kinase Nek6 \| Never in mitosis A-related kinase 6 \| Protein kinase SID6-1512 \| NimA-related protein kinase 6 \| NEK6

Background
NIMA‑related kinase 6 (NEK6) is a serine/threonine protein kinase of the NIMA family that functions in mitotic cell cycle control, where it is required for progression through metaphase, proper chromosome segregation, robust mitotic spindle formation, and completion of cytokinesis. The kinase has a mostly globular catalytic domain with a short, conformationally flexible N‑terminal extension and lacks large accessory regulatory domains, and it behaves as a monomeric enzyme that depends on upstream activating kinases rather than stable multiprotein complexes for its core activity. Activation of NEK6 occurs downstream of NEK9, which is itself activated by CDK1‑ and PLK1‑dependent phosphorylation and then phosphorylates NEK6 on defined serine residues, establishing a NEK9–NEK6 kinase cascade that operates during G2/M to promote mitotic entry and progression. NEK6 phosphorylates a range of substrates involved in spindle dynamics and chromosome movement, including the kinesin KIF11/EG5 and β‑tubulin, and these phosphorylation events support assembly and maintenance of a bipolar mitotic spindle and efficient chromosome congression. The kinase also targets regulatory and chromatin‑associated proteins such as histones H1 and H3, transcriptional regulators, and factors linked to translational control, integrating its activity with broader control of gene expression and cell cycle–related transcriptional programs. NEK6 participates in the DNA damage response by contributing to G2/M phase arrest after genotoxic stress, and inhibition or loss of NEK6 activity under these conditions is associated with mitotic failure and apoptosis, indicating that NEK6 activity is necessary for the survival of dividing cells under checkpoint control. Expression of NEK6 is elevated in several human cancers, and NEK6 activity associates with suppression of p53‑dependent senescence, linking this kinase to oncogenic pathways that favor continued proliferation and resistance to growth arrest. Across tumor contexts, NEK6 expression correlates with pathways such as PI3K–Akt, cell cycle progression, and actin cytoskeleton regulation, and NEK6‑high tumors show transcriptional signatures consistent with enhanced mitotic activity and altered cell adhesion and migration.

Background

NIMA‑related kinase 6 (NEK6) is a serine/threonine protein kinase of the NIMA family that functions in mitotic cell cycle control, where it is required for progression through metaphase, proper chromosome segregation, robust mitotic spindle formation, and completion of cytokinesis. The kinase has a mostly globular catalytic domain with a short, conformationally flexible N‑terminal extension and lacks large accessory regulatory domains, and it behaves as a monomeric enzyme that depends on upstream activating kinases rather than stable multiprotein complexes for its core activity. Activation of NEK6 occurs downstream of NEK9, which is itself activated by CDK1‑ and PLK1‑dependent phosphorylation and then phosphorylates NEK6 on defined serine residues, establishing a NEK9–NEK6 kinase cascade that operates during G2/M to promote mitotic entry and progression. NEK6 phosphorylates a range of substrates involved in spindle dynamics and chromosome movement, including the kinesin KIF11/EG5 and β‑tubulin, and these phosphorylation events support assembly and maintenance of a bipolar mitotic spindle and efficient chromosome congression. The kinase also targets regulatory and chromatin‑associated proteins such as histones H1 and H3, transcriptional regulators, and factors linked to translational control, integrating its activity with broader control of gene expression and cell cycle–related transcriptional programs. NEK6 participates in the DNA damage response by contributing to G2/M phase arrest after genotoxic stress, and inhibition or loss of NEK6 activity under these conditions is associated with mitotic failure and apoptosis, indicating that NEK6 activity is necessary for the survival of dividing cells under checkpoint control. Expression of NEK6 is elevated in several human cancers, and NEK6 activity associates with suppression of p53‑dependent senescence, linking this kinase to oncogenic pathways that favor continued proliferation and resistance to growth arrest. Across tumor contexts, NEK6 expression correlates with pathways such as PI3K–Akt, cell cycle progression, and actin cytoskeleton regulation, and NEK6‑high tumors show transcriptional signatures consistent with enhanced mitotic activity and altered cell adhesion and migration.

References
https://pubmed.ncbi.nlm.nih.gov/18728393/ https://pubmed.ncbi.nlm.nih.gov/19414596/

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%

NEK6 Antibody (Rabbit mAb) [P9K17]