Western blot analysis was performed on whole cell extracts (30 ug lysate) of Jurkat (Lane 1), MCF-7 (Lane 2), HEK-293 (Lane 3), NIH/3T3 (Lane 4), NTERA-2 (Lane 5) and HeLa (Lane 6). The blots were probed with Anti-Topoisomerase I Mouse Monoclonal Antibody (Product# 44321M, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 ug/ml, 1:2500 dilution). A ~ 90 kDa band corresponding to Topoisomerase I was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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 Pierce™ Power Blotter System (Product # 22834). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Peptide containing amino acid residues 699-725, conjugated to KLH.|
|Storage buffer||PBS, pH 7.3, with PEG, sucrose|
|Contains||0.09% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1000|
* 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.