Phospho-PLK1 (Thr210) Antibody [K10L6]

Application: Reactivity: Mouse, Human

Lane 1: Hela, Lane 2: Hela (thymidine, 2mM, 16 h; nocodazole, 10nM, 24 h), Lane 3: Hela (thymidine, 2mM, 16 h; nocodazole, 10nM, 24 h; phosphatase treated)

Usage Information

Dilution
WB1:1000 IHC1:500

Application
WB, IHC

Reactivity
Mouse, 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
68 kDa 68 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	Human colon tissue; Human placenta tissue; Human thyroid gland carcinoma tissue; Human gastroic carcinoma tissue; Human cervical carcinoma tissue; Mouse testis (RIPA); Mouse colon (RIPA); NIH/3T3 cells(RIPA); HeLa cells (calyculin A treated); Hela cells (thymidine, 2 mM, 16 h; nocodazole, 10nM, 24 h)
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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 ( recommending 5% BSA 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 RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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 ( recommending 5% BSA 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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
Phospho-PLK1 (Thr210) Antibody (Rabbit mAb) [K10L6] detects endogenous levels of total PLK1 protein only when it is phosphorylated at Thr210.

Subcellular Location
Centromere, Chromosome, Cytoplasm

Uniprot ID
P53350

Clone
K10L6

Synonym(s)
PLK, PLK1, Serine/threonine-protein kinase PLK1, Polo-like kinase 1, Serine/threonine-protein kinase 13, PLK-1, STPK13

Background
Phospho-PLK1 (Thr210) denotes the activated form of polo-like kinase 1 in which a threonine residue within the activation loop of the N‑terminal catalytic domain is phosphorylated, converting PLK1 from an autoinhibited state into a mitotic master kinase that coordinates multiple G2/M and mitotic processes including CDK1 activation, centrosome maturation, bipolar spindle assembly, chromosome segregation, and cytokinesis. PLK1 belongs to the CDC5/Polo subfamily of serine/threonine kinases and is organized into an N‑terminal kinase domain followed by a C‑terminal polo‑box domain that recognizes pre‑phosphorylated docking motifs on substrates and scaffolds, thereby conferring spatial precision and substrate selectivity once Thr210 phosphorylation has engaged the activation loop and relieved intramolecular inhibition. Aurora A kinase in complex with the cofactor Bora phosphorylates Thr210 at the G2/M transition through a CDK1-dependent switch, and this T‑loop phosphorylation is the principal event that generates catalytically active PLK1 at centrosomes and on mitotic structures, while dephosphorylation at this site returns PLK1 to a low-activity state. Active, Thr210‑phosphorylated PLK1 phosphorylates CDC25C and cyclin B1 to promote their nuclear accumulation and activation of CDK1, phosphorylates and inactivates inhibitory kinases such as PKMYT1/Myt1 and Wee1, and modifies components of the anaphase-promoting complex/cyclosome including Apc1, CDC16, and CDC27, thereby linking CDK1 activation, checkpoint satisfaction, and APC/C-dependent proteolysis into a coherent mitotic entry and exit program. PLK1 also phosphorylates a broad set of substrates at centrosomes, kinetochores, central spindle, and midbody, including NEDD1, KIZ, NINL, CEP55, PRC1, RACGAP1, BUB1B, SGOL1, STAG2, and CEP55, supporting centrosome maturation, attachment error correction, sister chromatid cohesion dynamics, spindle midzone organization, and completion of cytokinesis, and phospho‑Thr210 PLK1 shows characteristic enrichment at these structures during defined mitotic stages. Additional regulation of PLK1 activity superimposes on Thr210 phosphorylation through sites such as Ser99, which is phosphorylated downstream of PI3K–Akt to create a 14‑3‑3γ docking interface that further elevates PLK1 catalytic activity and is required for the timely metaphase–anaphase transition, indicating that Thr210 phosphorylation establishes a core active kinase state that can be modulated by parallel signaling inputs. Thr210 phospho‑status is sensitive to DNA damage signaling, with checkpoint activation suppressing T‑loop phosphorylation and PLK1 activity to prevent mitotic entry, whereas phospho‑mimetic Thr210 variants maintain kinase activity despite damage, highlighting phospho‑PLK1 (Thr210) as a direct indicator of checkpoint override or unscheduled mitosis in transformed cells. PLK1 expression and Thr210 phosphorylation levels are elevated in many solid tumors and hematologic malignancies and correlate with a high proliferative index and adverse prognosis.

Background

Phospho-PLK1 (Thr210) denotes the activated form of polo-like kinase 1 in which a threonine residue within the activation loop of the N‑terminal catalytic domain is phosphorylated, converting PLK1 from an autoinhibited state into a mitotic master kinase that coordinates multiple G2/M and mitotic processes including CDK1 activation, centrosome maturation, bipolar spindle assembly, chromosome segregation, and cytokinesis. PLK1 belongs to the CDC5/Polo subfamily of serine/threonine kinases and is organized into an N‑terminal kinase domain followed by a C‑terminal polo‑box domain that recognizes pre‑phosphorylated docking motifs on substrates and scaffolds, thereby conferring spatial precision and substrate selectivity once Thr210 phosphorylation has engaged the activation loop and relieved intramolecular inhibition. Aurora A kinase in complex with the cofactor Bora phosphorylates Thr210 at the G2/M transition through a CDK1-dependent switch, and this T‑loop phosphorylation is the principal event that generates catalytically active PLK1 at centrosomes and on mitotic structures, while dephosphorylation at this site returns PLK1 to a low-activity state. Active, Thr210‑phosphorylated PLK1 phosphorylates CDC25C and cyclin B1 to promote their nuclear accumulation and activation of CDK1, phosphorylates and inactivates inhibitory kinases such as PKMYT1/Myt1 and Wee1, and modifies components of the anaphase-promoting complex/cyclosome including Apc1, CDC16, and CDC27, thereby linking CDK1 activation, checkpoint satisfaction, and APC/C-dependent proteolysis into a coherent mitotic entry and exit program. PLK1 also phosphorylates a broad set of substrates at centrosomes, kinetochores, central spindle, and midbody, including NEDD1, KIZ, NINL, CEP55, PRC1, RACGAP1, BUB1B, SGOL1, STAG2, and CEP55, supporting centrosome maturation, attachment error correction, sister chromatid cohesion dynamics, spindle midzone organization, and completion of cytokinesis, and phospho‑Thr210 PLK1 shows characteristic enrichment at these structures during defined mitotic stages. Additional regulation of PLK1 activity superimposes on Thr210 phosphorylation through sites such as Ser99, which is phosphorylated downstream of PI3K–Akt to create a 14‑3‑3γ docking interface that further elevates PLK1 catalytic activity and is required for the timely metaphase–anaphase transition, indicating that Thr210 phosphorylation establishes a core active kinase state that can be modulated by parallel signaling inputs. Thr210 phospho‑status is sensitive to DNA damage signaling, with checkpoint activation suppressing T‑loop phosphorylation and PLK1 activity to prevent mitotic entry, whereas phospho‑mimetic Thr210 variants maintain kinase activity despite damage, highlighting phospho‑PLK1 (Thr210) as a direct indicator of checkpoint override or unscheduled mitosis in transformed cells. PLK1 expression and Thr210 phosphorylation levels are elevated in many solid tumors and hematologic malignancies and correlate with a high proliferative index and adverse prognosis.

References
https://pubmed.ncbi.nlm.nih.gov/23695676/ https://pubmed.ncbi.nlm.nih.gov/20869364/

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%

Phospho-PLK1 (Thr210) Antibody (Rabbit mAb) [K10L6]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support