Cyclin A1 + A2 Antibody (Rabbit mAb) [N12H18]

Application: Reactivity: Human

Lane 1: Hela, Lane 2: HepG2

Usage Information

Dilution
WB1:1000 IP1:70 IHC1:2000

Application
WB, IP, IHC

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
52 kDa 52 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 cancer tissue; Human testis tissue; Recombinant Human Cyclin A1 fragment; Full-length Human Cyclin A2 recombinant protein; HeLa cells; K562 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 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 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) , 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) , 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 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)，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) , 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) , 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 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
Cyclin A1 + A2 Antibody (Rabbit mAb) [N12H18] detects endogenous levels of total Cyclin A1 and A2 protein.

Subcellular Location
Nucleus, Cytoplasm

Uniprot ID
P78396, P20248

Clone
N12H18

Synonym(s)
Cyclin-A1 \| CCNA1 \| CCN1 \| CCNA \| CCNA2 \| Cyclin-A2 \| Cyclin-A \| Cyclin A

Background
Cyclin A1 and cyclin A2 are A‑type cyclins that form regulatory subunits of cyclin‑dependent kinases and together coordinate key transitions of the germline meiotic and somatic mitotic cell cycles, with cyclin A1 showing restricted expression in male germ cells and cyclin A2 displaying broad expression in proliferating somatic tissues. Both proteins share the characteristic cyclin box fold that provides the interface for binding CDKs, but they differ in expression timing and partner usage: cyclin A1 binds Cdk2 and Cdk1 in late pachytene and diplotene spermatocytes and is present around the first meiotic division, whereas cyclin A2 is abundant in spermatogonia and preleptotene spermatocytes and associates primarily with Cdk2 in germ cells, reflecting distinct functions in meiotic entry versus passage through meiosis. During male meiosis, cyclin A1 is required for progression from G2 into metaphase I; targeted disruption of the Ccna1 gene causes spermatogenic arrest before metaphase I, with defective activation of Cdc2 kinase and failure to generate active M‑phase‑promoting factor despite retention of B‑type cyclins, and cyclin A1–Cdc2/Cdk2 complexes can phosphorylate Cdc25A and Cdc25C phosphatases, indicating that cyclin A1 participates in an amplification loop that drives MPF activation and the G2/M transition in spermatocytes. Immunolocalization studies in testis show that cyclin A1 protein appears in germ cells just prior to or during the first meiotic division and then disappears, while cyclin A2 is expressed earlier in spermatogonia and preleptotene spermatocytes entering the meiotic pathway, and cyclin A1‑dependent kinase activity toward histone H1 is detectable in germ‑cell lysates, supporting a role for cyclin A1‑CDK complexes in chromatin condensation and meiotic chromosome segregation. In contrast, cyclin A2 is synthesized at the onset of S phase in somatic cells and binds Cdk2 to initiate and maintain DNA replication and prevent re‑replication by phosphorylating licensing factors such as CDC6 and MCM proteins, then later binds Cdk1 to promote G2/M progression and trigger activation of cyclin B1–Cdk1 complexes that drive chromatin condensation and nuclear envelope breakdown, making cyclin A2 a central regulator of both G1/S and G2/M transitions in the mitotic cycle. During mouse development and early embryogenesis, cyclin A2 is ubiquitously expressed in dividing cells and controls both DNA replication and spindle assembly, while cyclin A1 expression is restricted to spermatocytes, leukemia cells and certain post‑mitotic multiciliated cells, indicating that their differential expression patterns and CDK partner usage underpin specialization of A‑type cyclin function in germline versus somatic cell cycles. Genetic and functional work shows that cyclin A1 is essential for male fertility, with cyclin A1 haplo‑insufficiency reducing sperm count and quality, whereas cyclin A2 knockout is embryonic lethal due to failure of mitotic proliferation, underscoring that cyclin A1 is a critical meiotic regulator while cyclin A2 is indispensable for general cell division. Both cyclin A1 and A2 are implicated in tumor biology: cyclin A2 is frequently overexpressed in diverse cancers, where its elevated levels correlate with poor prognosis and may contribute to genomic instability through altered S‑phase and G2/M control, and cyclin A1 is expressed in certain leukemias and solid tumors, reflecting dysregulated germline cell‑cycle regulators in malignant contexts. A‑type cyclins A1 and A2 constitute a structurally related pair of CDK partners whose distinct expression domains and kinase interactions orchestrate G1/S and G2/M transitions across meiosis and mitosis, with cyclin A1 driving MPF activation and first meiotic division in male germ cells and cyclin A2 governing S‑phase entry, DNA replication fidelity and mitotic onset in somatic lineages, making this cyclin pair an informative target set for studies of cell‑cycle control, gametogenesis and cancer.

Background

Cyclin A1 and cyclin A2 are A‑type cyclins that form regulatory subunits of cyclin‑dependent kinases and together coordinate key transitions of the germline meiotic and somatic mitotic cell cycles, with cyclin A1 showing restricted expression in male germ cells and cyclin A2 displaying broad expression in proliferating somatic tissues. Both proteins share the characteristic cyclin box fold that provides the interface for binding CDKs, but they differ in expression timing and partner usage: cyclin A1 binds Cdk2 and Cdk1 in late pachytene and diplotene spermatocytes and is present around the first meiotic division, whereas cyclin A2 is abundant in spermatogonia and preleptotene spermatocytes and associates primarily with Cdk2 in germ cells, reflecting distinct functions in meiotic entry versus passage through meiosis. During male meiosis, cyclin A1 is required for progression from G2 into metaphase I; targeted disruption of the Ccna1 gene causes spermatogenic arrest before metaphase I, with defective activation of Cdc2 kinase and failure to generate active M‑phase‑promoting factor despite retention of B‑type cyclins, and cyclin A1–Cdc2/Cdk2 complexes can phosphorylate Cdc25A and Cdc25C phosphatases, indicating that cyclin A1 participates in an amplification loop that drives MPF activation and the G2/M transition in spermatocytes. Immunolocalization studies in testis show that cyclin A1 protein appears in germ cells just prior to or during the first meiotic division and then disappears, while cyclin A2 is expressed earlier in spermatogonia and preleptotene spermatocytes entering the meiotic pathway, and cyclin A1‑dependent kinase activity toward histone H1 is detectable in germ‑cell lysates, supporting a role for cyclin A1‑CDK complexes in chromatin condensation and meiotic chromosome segregation. In contrast, cyclin A2 is synthesized at the onset of S phase in somatic cells and binds Cdk2 to initiate and maintain DNA replication and prevent re‑replication by phosphorylating licensing factors such as CDC6 and MCM proteins, then later binds Cdk1 to promote G2/M progression and trigger activation of cyclin B1–Cdk1 complexes that drive chromatin condensation and nuclear envelope breakdown, making cyclin A2 a central regulator of both G1/S and G2/M transitions in the mitotic cycle. During mouse development and early embryogenesis, cyclin A2 is ubiquitously expressed in dividing cells and controls both DNA replication and spindle assembly, while cyclin A1 expression is restricted to spermatocytes, leukemia cells and certain post‑mitotic multiciliated cells, indicating that their differential expression patterns and CDK partner usage underpin specialization of A‑type cyclin function in germline versus somatic cell cycles. Genetic and functional work shows that cyclin A1 is essential for male fertility, with cyclin A1 haplo‑insufficiency reducing sperm count and quality, whereas cyclin A2 knockout is embryonic lethal due to failure of mitotic proliferation, underscoring that cyclin A1 is a critical meiotic regulator while cyclin A2 is indispensable for general cell division. Both cyclin A1 and A2 are implicated in tumor biology: cyclin A2 is frequently overexpressed in diverse cancers, where its elevated levels correlate with poor prognosis and may contribute to genomic instability through altered S‑phase and G2/M control, and cyclin A1 is expressed in certain leukemias and solid tumors, reflecting dysregulated germline cell‑cycle regulators in malignant contexts. A‑type cyclins A1 and A2 constitute a structurally related pair of CDK partners whose distinct expression domains and kinase interactions orchestrate G1/S and G2/M transitions across meiosis and mitosis, with cyclin A1 driving MPF activation and first meiotic division in male germ cells and cyclin A2 governing S‑phase entry, DNA replication fidelity and mitotic onset in somatic lineages, making this cyclin pair an informative target set for studies of cell‑cycle control, gametogenesis and cancer.

References
https://pubmed.ncbi.nlm.nih.gov/21630154/ https://pubmed.ncbi.nlm.nih.gov/10068472/

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%