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Anti-cdc2 Mouse Antibody [C23A20]

Catalog No.: F3545

Application: Reactivity: Human, Monkey

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:100 IF1:200 - 1:800

Application
WB, IP, IF

Reactivity
Human, Monkey

Source
Mouse

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
34 kDa

Positive Control	HeLa cell (G0, G1S, G2, M stages of cell cycle)
Negative Control

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. 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) and put the sample on ice for 5 min. 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) and put the sample on ice for 5 min. 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 5% skim milk powder 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.

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.

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) and put the sample on ice for 5 min.

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) and put the sample on ice for 5 min.

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 5% skim milk powder 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： Specimen Preparation 1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS. NOTE: Paraformaldehyde is toxic, use only in a fume hood. 2. Fix cells for 15 min at room temperature. 3. Aspirate fixative, rinse three times in 1X PBS for 5 min each. 4. Proceed with Immunostaining. Immunostaining 1. Add theblocking buffer and incubate for 60 min at RT. 2. Prepare primary antibody diluent in antibody dilution buffer as recommended . 3. Aspirate blocking solution, apply diluted primary antibody. 4. Incubate overnight at 4°C. 5. Rinse three times in 1X PBS for 5 min each. 6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark. 7. Rinse three times in 1X PBS for 5 min each. 8. Mount slides usingmounting medium with DAPI and cover with coverslips. 9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.

Experimental Protocol：

Specimen Preparation

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.

NOTE: Paraformaldehyde is toxic, use only in a fume hood.

2. Fix cells for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.

2. Prepare primary antibody diluent in antibody dilution buffer as recommended .

3. Aspirate blocking solution, apply diluted primary antibody.

4. Incubate overnight at 4°C.

5. Rinse three times in 1X PBS for 5 min each.

6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.

7. Rinse three times in 1X PBS for 5 min each.

8. Mount slides usingmounting medium with DAPI and cover with coverslips.

9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.

Datasheet & SDS

Biological Description

Specificity
Anti-cdc2 Mouse Antibody [C23A20] recognizes endogenous levels of total cdc2 protein.

Subcellular Location
Cytoplasm, Cytoskeleton, Mitochondrion, Nucleus

Uniprot ID
P06493

Clone
C23A20

Synonym(s)
Cyclin-dependent kinase 1; CDK1; Cell division control protein 2 homolog; Cell division protein kinase 1; p34 protein kinase; CDC2; CDC28A; CDKN1; P34CDC2

Background
Cdc2, also known as CDK1 (Cyclin-Dependent Kinase 1), is a serine/threonine kinase that is indispensable for eukaryotic cell cycle progression, particularly the G2–M transition. Cdc2 contains a catalytic domain with an ATP-binding site and a regulatory T-loop, which undergoes conformational changes upon binding to cyclins, most notably cyclin B, forming the maturation-promoting factor (MPF). Cdc2 activity is tightly regulated by phosphorylation: it is activated by CAK-mediated phosphorylation at Thr161 and inhibited by Wee1/Myt1-mediated phosphorylation at Thr14 and Tyr15, with Cdc25 phosphatases reversing these inhibitory modifications to trigger mitotic entry. Cdc2 can compensate for CDK2, CDK4, and CDK6, driving the G1–S transition in their absence, and phosphorylates pRb to release E2F and activate cell cycle gene transcription. It also regulates centrosome separation, Golgi fragmentation, spindle assembly, and interacts with CDK inhibitors p21^Cip1 and p27^Kip1, as well as transcription factors such as FOXO1 and RUNX2, thereby linking cell cycle control to apoptosis and differentiation. Cdc2 expression is regulated by E2F family transcription factors and suppressed by Rb-E2F complexes, while its overexpression or hyperactivation is associated with tumorigenesis, genomic instability, and resistance to cell cycle checkpoints.

Background

Cdc2, also known as CDK1 (Cyclin-Dependent Kinase 1), is a serine/threonine kinase that is indispensable for eukaryotic cell cycle progression, particularly the G2–M transition. Cdc2 contains a catalytic domain with an ATP-binding site and a regulatory T-loop, which undergoes conformational changes upon binding to cyclins, most notably cyclin B, forming the maturation-promoting factor (MPF). Cdc2 activity is tightly regulated by phosphorylation: it is activated by CAK-mediated phosphorylation at Thr161 and inhibited by Wee1/Myt1-mediated phosphorylation at Thr14 and Tyr15, with Cdc25 phosphatases reversing these inhibitory modifications to trigger mitotic entry. Cdc2 can compensate for CDK2, CDK4, and CDK6, driving the G1–S transition in their absence, and phosphorylates pRb to release E2F and activate cell cycle gene transcription. It also regulates centrosome separation, Golgi fragmentation, spindle assembly, and interacts with CDK inhibitors p21^Cip1 and p27^Kip1, as well as transcription factors such as FOXO1 and RUNX2, thereby linking cell cycle control to apoptosis and differentiation. Cdc2 expression is regulated by E2F family transcription factors and suppressed by Rb-E2F complexes, while its overexpression or hyperactivation is associated with tumorigenesis, genomic instability, and resistance to cell cycle checkpoints.

References
https://pubmed.ncbi.nlm.nih.gov/21535261/ https://pubmed.ncbi.nlm.nih.gov/15888452/

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
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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%