Choose Your Country or Region

Phospho-CDK2 (Thr 160) Rabbit mAb

Catalog No.: F3552

Lane 1: C6
Lane 2: C6 (Nocodazole, 50 ng/ml, 37℃,20 h)

Experiment Essentials

Subcellular Location: Cytoplasm, Cytoskeleton, Endosome, Nucleus.
WB
Recommending using RIPA/NP-40 Lysis Buffer to prepare lysates.
Recommended primary antibody dilution ratio 1:2000.

Usage Information

Dilution
WB1:2000-1:6000 IHC1:50 - 1:200 IF1:50 - 1:200

Application
WB, IHC, IF, ELISA

Source
Rabbit

Reactivity
Human, Mouse, Rat

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
34kDa

Exprimental 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)，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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 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.Add an appropriate volume of RIPA/NP-40 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 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), 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 1389. 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 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)，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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 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.Add an appropriate volume of RIPA/NP-40 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 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), 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

1389. 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
Phospho-CDK2 (Thr 160) Rabbit mAb recognizes endogenous levels of CDK2 only when phosphorylated at Thr 160.

Clone
N24F24

Background
The phosphorylation of CDK2 at threonine 160 (Thr160) is a crucial event for its full activation as a cyclin-dependent kinase, allowing it to regulate cell cycle progression. Thr160 is located in the activation loop (AL) of CDK2, and its phosphorylation stabilizes the AL, aligning substrates for efficient catalysis by reducing its flexibility. The phosphorylation at this site is typically mediated by CDK-activating kinase (CAK), which transfers a phosphate group from ATP to Thr160. This phosphorylation enables the formation of ionic interactions with a lysine residue at the P+3 position of substrates, promoting substrate recognition and enhancing the catalytic efficiency of CDK2. CDK2 operates as part of complexes with cyclins (such as Cyclin A and Cyclin E), and phosphorylation at Thr160 significantly boosts its enzymatic activity in these complexes. The enzyme catalyzes phosphoryl transfer reactions with the help of Mg²⁺ ions in the active site, stabilizing transition states during catalysis. Dephosphorylation of Thr160 by phosphatases, like KAP, inhibits CDK2 activity unless bound to cyclins, ensuring proper regulation. CDK2 can also undergo autophosphorylation at Thr160 under specific conditions, further modulating its activation. This phosphorylation event is tightly regulated during the cell cycle, peaking during the S phase and G2 phase to ensure proper progression through the cell cycle. Disruption of Thr160 phosphorylation impairs substrate recognition and enzymatic activity, which can hinder proper cell cycle regulation.

Background

The phosphorylation of CDK2 at threonine 160 (Thr160) is a crucial event for its full activation as a cyclin-dependent kinase, allowing it to regulate cell cycle progression. Thr160 is located in the activation loop (AL) of CDK2, and its phosphorylation stabilizes the AL, aligning substrates for efficient catalysis by reducing its flexibility. The phosphorylation at this site is typically mediated by CDK-activating kinase (CAK), which transfers a phosphate group from ATP to Thr160. This phosphorylation enables the formation of ionic interactions with a lysine residue at the P+3 position of substrates, promoting substrate recognition and enhancing the catalytic efficiency of CDK2. CDK2 operates as part of complexes with cyclins (such as Cyclin A and Cyclin E), and phosphorylation at Thr160 significantly boosts its enzymatic activity in these complexes. The enzyme catalyzes phosphoryl transfer reactions with the help of Mg²⁺ ions in the active site, stabilizing transition states during catalysis. Dephosphorylation of Thr160 by phosphatases, like KAP, inhibits CDK2 activity unless bound to cyclins, ensuring proper regulation. CDK2 can also undergo autophosphorylation at Thr160 under specific conditions, further modulating its activation. This phosphorylation event is tightly regulated during the cell cycle, peaking during the S phase and G2 phase to ensure proper progression through the cell cycle. Disruption of Thr160 phosphorylation impairs substrate recognition and enzymatic activity, which can hinder proper cell cycle regulation.

References
[1] De Vivo M, et al. Proteins. 2006 Jan 1;62(1):89-98. [2] Holmes JK, et al. Eur J Biochem. 2001 Sep;268(17):4647-52.

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

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%