Western blot analysis was performed on nuclear enriched extracts (30 ug lysate) of HCT 116 (Lane 1), Hep G2 (Lane 2), Caco-2 (Lane 3) and PC-3 (Lane 4).The blots were probed with TOP1 Mouse monoclonal Antibody (Product# 435900, 2 ug/ml) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 ug/ml, 1:4000 dilution). A 100 kDa band corresponding to TOP1 was observed across the cell lines tested. Apart from desired band, a 70 kDa band was observed in Hep G2 cell line representing the C terminal fragment of the enzyme. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Recombinant protein derived from the N-terminal region of human TOP1 protein.|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|Immunofluorescence (IF)||Assay Dependent|
|Immunoprecipitation (IP)||Assay Dependent|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Topoisomerases are nuclear enzymes involved in a variety of cellular activities such as chromosome condensation, DNA replication, transcription, recombination and segregation at mitosis. Human topoisomerase I is a 100kDa protein capable of relaxing positively and negatively supercoiled DNA by performing a transient single stranded nick which is then relegated at the end of the reaction. It has been shown that the enzyme is located in regions of the genome that are undergoing active RNA synthesis, where it probably reduces superhelical stresses in the DNA, enabling RNA polymerase to function properly. Both DNA topoisomerases I and II have been found to be targets of autoantibodies in the sera of patients with certain autoimmune diseases such as systemic lupus erythematosus, and also of some anti tumor drugs and antibiotics. Elevated levels of DNA topoisomerase I, detected by transfer assays, have been demonstrated in colorectal tumors compared with normal colon mucosa as a result of increased transcription or mRNA stability.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.