MaxLight™550 is a new Yellow-Green photostable dye conjugate comparable to Alexa Fluor™546, 555, DyLight™549 , Cy3™, TRITC and offers better labeling efficiency, brighter imaging and increased immunodetection. Absorbance (550nm); Emission (575nm); Extinction Coefficient 150,000.
Recognizes a protein of 62kD, identified as cdc6. The replication licensing system acts to ensure that no section of the genome is replicated more than once in a single cell cycle. The origin recognition complex (ORC) and cdc6/cdc18 are needed on chromatin before the licensing reaction can take place. During the transition from a growth-arrested to a proliferative state, transcription of mammalian cdc6 is regulated by the cell cycle transcription factor (E2F protein). Immunoblots show that quiescent cells lack cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum cdc6 accumulation and MCM protein binding to chromatin in vivo. Antibodies against cdc6 and MCM5 stain abnormal (neoplastically transformed) cells in cervical smears and sections with remarkably high specificity and sensitivity. Thus antibodies against proteins that regulate DNA replication may reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.
Suitable for use in Immunoblot and Immunoprecipitation.
Immunoblot: 1–4ug/ml detects cdc6 in HeLa nuclear extract and 3T3 nuclear extract. HeLa nuclear extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-cdc6 (1ug/ml). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system.
Immunoprecipitation: To be determined by the researcher.
Positive Antigen Control:
HeLa nuclear extract. Use 20ug per lane for minigels.
1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a nuclear extract and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.
2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 60 minutes at 20-25°C with constant agitation.
3. Incubate the nitrocellulose with 1-4ug/ml of anti-cdc6, diluted in freshly prepared PBS-MLK, for 2 hours with agitation at room temperature.
4. Wash the nitrocellulose twice with water.
5. Incubate the nitrocellulose in the secondary reagent of choice ( a goat anti-mouse HRP conjugated IgG, 1:3000 dilution was used) in PBS-MLK for 1.5 hours at room temperature with agitation.
6. Wash the nitrocellulose with water twice.
7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 15 minutes.
8. Rinse the nitrocellulose in 4-5 changes of water for 2 hours.
9. Use detection method of choice (enhanced chemiluminescence was used).
Storage and Stability:
May be stored at 4°C for short-term only. For long-term storage and to avoid repeated freezing and thawing, add glycerol (25–50%) and freeze at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Note: Applications are based on unconjugated antibody.