Cyclin-Dependent Kinase 4 (CDK4) antibody

Details for Product No. ABIN967650
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Antigen
Synonyms zgc:153726, CMM3, PSK-J3, Crk3, 8-6, CDK4, CDK4/6, CG5072, Cdk4/6, DmCdk4, Dmel\\CG5072, Pk53C, Pk?7, cdk, cdk4, cdk4/6, dCdk4, l(2)05428, l(2)0671, l(2)k06503, l(2)s4639, l(2)sh0671, xcdk4
Reactivity
Human
(175), (65), (50), (20), (14), (5), (1)
Host
Mouse
(100), (84), (5), (2), (1)
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
(7), (5), (5), (3), (3), (3), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1)
Application
Western Blotting (WB), Immunoprecipitation (IP), BioImaging (BI), Immunohistochemistry (Frozen Sections) (IHC (fro))
(175), (62), (54), (39), (28), (17), (15), (10), (10), (9), (8), (5), (2), (2), (1)
Pubmed 3 references available
Quantity 0.1 mg
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Catalog No. ABIN967650
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Immunogen Human Cdk4
Clone DCS-35
Isotype IgG1
Cross-Reactivity Dog (Canine), Rat (Rattus), Mouse (Murine)
Characteristics 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Triton is a trademark of the Dow Chemical Company.
Purification Purified from tissue culture supernatant or ascites by affinity chromatography.
Alternative Name Cdk4
Background Cyclins, cyclin-dependent kinases (Cdks), and cyclin-dependent kinase inhibitors (CdkIs) are essential for cell-cycle control in eukaryotes (reviewed in 1). Cyclins, regulatory subunits, bind to cyclin-dependent kinases (Cdks), catalytic subunits, to form active cyclin-Cdk complexes. Cdk subunits by themselves are inactive and binding to a cyclin is required for their activity. Cyclins A, B1, D and E undergo periodic synthesis and degradation, thereby providing a mechanism to regulate Cdk activity throughout the cell cycle. Additionally, Cdk activity is further regulated by activating and inhibitory phosphorylations, and small proteins (p15, p16, p18, p19, p21 and p27), called CdkIs, that bind to cyclins, Cdks, or cyclin-Cdk complexes. Cdk4 was originally called PSK-J3,2 and following demonstration of its association with D-type cyclins, became known as Cdk4.2 D-type cyclins also associate with Cdks 2 and 5, although Cdk4 appears to be the most abundant partner. The D-type cyclins (D1, D2, and D3) are expressed in response to growth factors or mitogens, and rapidly degrade when mitogens are withdrawn. D cyclins appear to promote G0 to G1 transitions and the rate of G1 progression. For example, cyclin D-Cdk4 and cyclin D-Cdk6 complexes phosphorylate the retinoblastoma protein (Rb) which removes the G1 phase block caused by underphosphorylated Rb. Cdk4 has a molecular weight of ~33 kD.
Molecular Weight 33 kDa
Research Area Cancer, Cell Cycle, Kinases/Phosphatases
Application Notes Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in an 96-well Imaging Plate and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 myl of Fixation Buffer to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either 90% methanol, or Triton™ X-100: a. Add 100 myl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. OR b. Add 100 myl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 myl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 myl of to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 myl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 myl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 myl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 myl per well of 2 myg/ml Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Comment

Related Products: ABIN967389

Restrictions For Research Use only
Format Liquid
Concentration 0.5 mg/ml
Buffer Aqueous buffered solution.
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage 4 °C
Supplier Images
anti-Cyclin-Dependent Kinase 4 (CDK4) antibody Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate at ~10 000 cells per well. After overnight incubation, cells were stained using the alcohol perm protocol and the anti-Cdk4 antibody. The second step reagent was Alexa Fluor® 555 (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager system using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols.
anti-Cyclin-Dependent Kinase 4 (CDK4) antibody (2) Western blot analysis of Cdk4. 293 cell lysates were probed with 5 µg/ml (lane 1), 2 µg/ml (lane 2) or 0.5 µg/ml (lane 3) of anti-Cdk4 (clone DCS-35). The antibody identifies Cdk4 at ~33 kDa.
anti-Cyclin-Dependent Kinase 4 (CDK4) antibody (3) anti-Cyclin-Dependent Kinase 4 (CDK4) antibody (Image 3)
Product cited in: Matsushime, Ewen, Strom et al.: "Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins." in: Cell, Vol. 71, Issue 2, pp. 323-34, 1992 (PubMed).

Depoortere, Van Keymeulen, Lukas et al.: "A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes." in: The Journal of cell biology, Vol. 140, Issue 6, pp. 1427-39, 1998 (PubMed).

Johnson, Walker: "Cyclins and cell cycle checkpoints." in: Annual review of pharmacology and toxicology, Vol. 39, pp. 295-312, 1999 (PubMed).

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