Anti-Topoisomerase I antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Topoisomerase I antibody (AB85038)

ICC/IF image of ab85038 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab85038, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 100% methnaol fixed (5 min) HeLa cells at 1µg/ml.

• IP

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Immunoprecipitation - Anti-Topoisomerase I antibody (AB85038)

Topoisomerase I was immunoprecipitated using 0.5mg Hek293 whole cell extract, 5µg of Rabbit polyclonal to Topoisomerase I and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hek293 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab85038.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 90kDa; Topoisomerase I

All lanes:

Immunoprecipitation - Anti-Topoisomerase I antibody (ab85038)

Predicted band size: 91 kDa

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• WB

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Western blot - Anti-Topoisomerase I antibody (AB85038)

Human DNA topoisomerase 1 contains a potential sumoylation site (SwissProt) which may explain its migration at a higher molecular weight than predicted.

All lanes:

Western blot - Anti-Topoisomerase I antibody (ab85038) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 5:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 91 kDa

Observed band size: 90 kDa

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Exposure time: 30s

• WB

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Western blot - Anti-Topoisomerase I antibody (AB85038)

All lanes:

Western blot - Anti-Topoisomerase I antibody (ab85038) at 1 µg/mL

Lane 1:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 2:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 91 kDa

Observed band size: 100 kDa,50 kDa,55 kDa,70 kDa

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Exposure time: 3min