CDK4 antibody - C-terminal region (ARP30259_P050)

Catalog#: ARP30259_P050

• Size: 50ug

Human HepG2

Human HCT116

Human HCT116

• Description of Target: Cdk4 is probably involved in the control of the cell cycle.
• Gene Symbol: CDK4
• Official Gene Full Name: Cyclin-dependent kinase 4
• Alias Symbols: CMM3; MGC14458; PSK-J3
• Protein Accession#: NP_000066
• Nucleotide Accession#: NM_000075
• Swissprot Id: P11802
• Host: Rabbit
• Protein Size (# AA): 303
• Molecular Weight: 34kDa
• Application: WB
• Partner Proteins: MYC,BRCA1,CCND1,CCND1,CCND2,CDKN1A,CDKN2A,CDKN2A,CDKN2A,CDKN2A,CDKN2C,RB1,SMAD2,SMAD3,tax,BAG6,BIRC5,BMPR1B,BRCA1,CAPNS1,CCND1,CCND2,CCND3,CDC37,CDC45,CDC7,CDKN1A,CDKN1B,CDKN1C,CDKN2A,CDKN2B,CDKN2C,CDKN2D,CEBPA,CNOT7,IFI27,MCM2,MYOD1,NCOA2,NOL12,ORC3,PPP2R1B,PRKAR1A,PSMD10,PTMA,RB1,RBL1,RFC1,RFC4,SERTAD1,SETDB1,SMAD2,SMAD3,TGFBR1,VTA1,ZNF655,ACTR3B,BAG6,BIRC5,BRCA1,CAPNS1,CCND1,CCND2,CCND3,CDC37,CDKN1A,CDKN1B,CDKN2A,CDKN2B,CDKN2C,CDKN2D,CDT1,CEBPA,CNOT7,DBNL,GEMIN4,HSP82,HSPA8,IME2,LYN,MYOD1,NCOA2,PCNA,PRMT5,RB1,RFC1,RPRM,SAP25,SERTAD1,SETDB1,SMC1A,SMC3,SP1,TSPYL2,UBXN1,VTA1,WDR77
• Product Format: Lyophilized powder
• Predicted Homology Based on Immunogen Sequence: Human: 100%; Pig: 91%; Dog: 83%; Rabbit: 83%; Rat: 75%
• Peptide Sequence: Synthetic peptide located within the following region: PRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE
• Blocking Peptide: For anti-CDK4 antibody is Catalog # AAP30259
• Key Reference: N/A
• Reconstitution and Storage: Add 50 μl of distilled water. Final anti-CDK4 antibody concentration is 1 mg/ml in PBS buffer. For longer periods of storage, store at -20°C. Avoid repeat freeze-thaw cycles.

Product Review: CDK4 antibody - C-terminal region (ARP30259_P050) in Human colon carcinoma cell line HCT116 using Western Blot

Product Page for CDK4 antibody - C-terminal region (ARP30259_P050)

Researcher: Anke Rauch & Dr. Oliver Krämer, Institute for Biochemistry and Biophysics, Friedrich-schiller University Jena
Application: Western Blotting
Species+tissue/cell type: Human colon carcinoma cell line HCT116
How many ug’s of tissue/cell lysate run on the gel:
1. 30 ug human HCT116 cell lysate
2. 30 ug genotoxic treated human HCT116 cell lysate
3. 30 ug genotoxic treated human HCT116 cell lysate
Primary antibody dilution: 1:2000
Secondary antibody: Anti-rabbit HRP
Secondary antibody dilution: 1:5000

Questionnaire:
How do Aviva’s reagents play a role in your experimental goals?	The Aviva antibody against CDK4 is used to investigate the influence of genotoxic agents on cell cycle regulation and survival. In this contest protein levels of CDK4 shall give information about the differential induction of cell cycle arrest upon genotoxic stress in human colon carcinoma cells.
How would you rate this antibody on a scale from 1-5 (5=best) and why?	Antibody is rated at 5. Signals are precise, without unspecific bands.
Would you use this antibody in future experiments?	Yes.
Have you used another antibody which has worked in your application?	No.
Do you believe the information about the reagent on Aviva’s website is correct?	The antibody was applied in a recommended concentration of 0.5 ug/ml and principly worked. Nevertheless the immunoblot presented on the datasheet showed nearly no unspecific bands.
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?	The antibody shall be used in further experiements and data are planned to be published.
How did you store the antibody after re-suspension?	Resuspended in distilled water, stored at – 20 degree C.
Sample Description, Species, Tissue/Cell Type:	Human colon carcinoma cell line HCT116, 30ug protein per lane has been loaded.
How many different experimental trials were conducted using the antibody sample?	Two experimental trials.
What type of experimental sample are you using and how did you preparing it?	Cells were harvested from cell culture plates by scraping. Cell suspension was transferred into 1.5ml tubes and  centrifuged for 2 minutes at 700g. After discarding the supernatant cells were incubated with 150 ul lysis buffer (containing 10 % (v/v) Glycerol, 0.5% (v/v) NP-40, 0.1% (v/v) NaF, 1% (v/v) NaV and 0.2 % protease inhibitor cocktail) on ice. Subsequently cells were sonified with 30 % amplitude for 5 seconds. Samples were centrifuged for 10 minute at 14000 rpm and supernatant was used for westernblot analysis.
Primary used and dilution:	Primary antibody was diluted 1:2000 and applied for 15 hours.
Secondary used and dilution:	Secondary antibody against rabbit-IgG (Santa Cruz) was appliedin a 1:5000 dilution and incubated for 1 hour.
What controls were used in your experiment? Please include your positive control:	Untreated cells were used as a negative control for genotoxic stress.
Experimental Procedure/Protocols:	Proteins were blotted on a PVDF membrane. Thereafter the membrane was incubated in 5% milk and PBS-T (PBS + 0.05% Tween20 (Roth)) at room temperature for 1 h to block all aunspecific protein binding sites. Specific primary and secondary antibodies were used for protein detection. Primary antibodies were dilluted in 2% milk PBS-T and overnight at 4 degree C on a roll mixer. After incubation the membrane was washed 3 times with 15ml PBS-T for 10 minutes and specific secondary antibody against rabbit immunogloulins was applied for 1 h at room temperature. The membrane was washed 3 times for 10 minutes in PBS-T to rinse off the extra secondary antibodies. The membrane was transferred onto a plastic bag and incubated with SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific (1:1) for 2 minutes before it was developed.