Cyclin D1 (C-terminal) Antibody [A1D5]

N. catalogo F2524

Stampa

Descrizione biologica

Specificità

Cyclin D1 (C-terminal) Antibody [A1D5] rileva i livelli endogeni totali della proteina Cyclin D1.
Contesto

La Cyclin D1, un regolatore chiave del Cell Cycle, svolge un ruolo fondamentale nello sviluppo del cancro guidando la proliferazione cellulare incontrollata. È una proteina di 36 kDa codificata dal gene CCND1, situato sul cromosoma 11q13. Strutturalmente, la Cyclin D1 contiene un dominio di legame alla proteina RB, una regione per il legame delle CDK o dei loro inibitori, un motivo LxxLL per il reclutamento dei coattivatori, una sequenza PEST per la degradazione e un residuo di treonina critico che regola l'esportazione nucleare e la stabilità delle proteine. La Cyclin D1 è espressa nella maggior parte delle cellule umane normali, ad eccezione di quelle derivate da linee di cellule staminali del midollo osseo. La sua espressione è rapidamente indotta da fattori di crescita, consentendole di integrare e regolare molteplici vie di segnalazione intracellulari che controllano la proliferazione cellulare. La disregolazione dell'espressione, dell'accumulo, dell'ubiquitinazione e dell'assemblaggio della Cyclin D1 con le sue CDK affini porta a una crescita cellulare eccessiva, posizionando la Cyclin D1 come un driver oncogenico in tumori come il cancro al seno, il cancro al polmone e il melanoma. Mentre la sua espressione è strettamente regolata nelle cellule normali, le cellule tumorali sfruttano vari meccanismi per amplificare l'attività della Cyclin D1. La Cyclin D1 facilita la transizione dalla fase G1 alla fase S agendo come un regolatore allosterico di CDK4 e CDK6. Il complesso attivo Cyclin D1/CDK4 si sposta nel nucleo, dove fosforila la proteina del retinoblastoma (RB), in congiunzione con Cyclin E/CDK2. Questa fosforilazione rilascia la repressione mediata da RB dei fattori di trascrizione E2F, consentendo la trascrizione di geni essenziali per la proliferazione cellulare. Livelli elevati di Cyclin D1 guidano la proliferazione incontrollata e la crescita tumorale, sottolineando il suo ruolo centrale nella patogenesi del cancro. Oltre alle sue funzioni canoniche dipendenti da CDK, la Cyclin D1 mostra attività indipendenti da CDK, agendo come modulatore trascrizionale. Influenze il controllo del Cell Cycle e la proliferazione interagendo con vari fattori di trascrizione. La Cyclin D1 interagisce anche con recettori nucleari come ERα, AR, il recettore gamma attivato dai proliferatori di perossisomi (PPARγ), il recettore beta dell'ormone tiroideo (TR-β) e i loro coregolatori, influenzando il Cell Cycle, la crescita e la differenziazione. Inoltre, governa l'espressione di microRNA (miRNA) specifici e contribuisce significativamente alle interazioni tumore-stroma, amplificando varie caratteristiche del cancro. I ruoli multifaccettati della Cyclin D1 nell'inizio e nella progressione del cancro riflettono la sua complessità come regolatore della funzione cellulare sia dipendente che indipendente da CDK. 

Informazioni sullutilizzo

Applicazione WB, IP, IHC, IF Diluizione
WB IP IHC IF
1:10000 -1: 50000 1:30 1:100 - 1:500 1:50
Reattività Human, Mouse, Rat
Fonte Rabbit Monoclonal Antibody MW 34 kDa
Tampone di conservazione PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃
Conservazione
(Dalla data di ricevimento)		 -20°C (avoid freeze-thaw cycles), 2 years
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, 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 1:10000), 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
1238. 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:
 
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
 
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
 
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
Wash the sample with PBS for 3 times, 3 minutes each time.
 
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
 
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
 
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
 
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.
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.
 

Riferimenti

  • https://pubmed.ncbi.nlm.nih.gov/33317149/

Dati di applicazione

WB

Validato da Selleck

  • F2524-wb
    Lane 1: A549
    Lane 2: A549 (KO CCND1)
    Lane 3: SH-SY5Y
    Lane 4: Mouse kidney
    Lane 5: Mouse spleen
    Lane 6: Rat heart

IF

Validato da Selleck

  • F2524-IF
    Immunofluorescent analysis of MCF-7 cells using F2524 (green, 1:50), Hoechst (blue) and tubulin (Red).