CDT1 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Geminin is a protein that inhibits DNA replication by preventing the loading of MCM complex on chromatin. Human CDT1 is a 65kDa protein that contains 546 amino acids and coimmunoprecipitates with Geminin. Inhibition of DNA replication by Geminin in cell-free cDNA replication extracts can be reversed by the addition of excess CDT1. In the normal cell cycle, CDT1 is present only in G1 and S phases, whereas Geminin is present in S and G2 phases. Wohlschlegel et al. (2000) concluded that their results suggest that Geminin inhibits inappropriate origin firing by targeting CDT1. This antibody was raised by a genetic immunization technique. Genetic immunization can be used to generate antibodies by directly delivering antigen-coding DNA into the animal, rather than injecting a protein or peptide (Tang et al. PubMed: 1545867; Chambers and Johnston PubMed 12910245; Barry and Johnston PubMed: 9234514). The animals cells produce the protein, which stimulates the animals immune system to produce antibodies against that particular protein. A vector coding for a partial fusion protein was used for genetic immunisation of a mouse and the resulting serum was tested in Western blot against an E.coli lysate containing that partial fusion protein. Genetic immunization offers enormous advantages over the traditional protein-based immunization method. DNA is faster, cheaper and easier to produce and can be produced by standard techniques readily amenable to automation. Furthermore, the antibodies generated by genetic immunization are usually of superior quality with regard to specificity, affinity and recognizing the native protein.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-09063

• CDT1 Antibodies
• CDT1 Lysate