Analysis of replicating DNA were performed according to the methods described previously32 (link). In brief, cells were pulse-labeled with 50 μM CldU for 15 min, followed by second labeling with 50 μM IdU for 15 min before analysis. Cells were harvested and DNA fibers were stretched onto glass slides in a DNA lysis buffer (200 mM Tris-HCl at pH 7.4, 0.5% SDS, 50 mM EDTA). Single replicating DNA was detected with rat anti-CldU (Novus), mouse anti-IdU (Becton Dickinson) and anti-single-strand DNA (EMD Millipore) antibodies. Images were captured using the Nikon Eclipse TiE microscope and processed using NIS Elements AR imaging software.
Rat anti cldu
Manufactured by Novus Biologicals
Rat anti-CldU is a primary antibody that specifically recognizes and binds to the synthetic nucleoside analog 5-chloro-2'-deoxyuridine (CldU). This antibody can be used to detect and label cells that have incorporated CldU during DNA synthesis, providing a means to analyze cell proliferation and DNA replication dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti e microscope, by National Instruments (2 mentions)
Mouse anti idu, by BD (2 mentions)
Anti single strand dna, by Merck Group (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Goat anti rat alexafluor 594, by Thermo Fisher Scientific (1 mentions)
Mouse anti brdu, by BD (1 mentions)
3 protocols using rat anti cldu
1
Replicating DNA Analysis Protocol
2
Replicating DNA Analysis Protocol
3
Dual-Pulse Labeling for DNA Replication Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Unsynchronized cells were pulse-labeled for 30 min by a medium containing 100 μM of the thymidine analog iododeoxyuridine (IdU). At the end of the first labeling period, the cells were washed twice with a warm medium and pulse-labeled once more for 30 min with a medium containing 100 μM of another thymidine analog chlorodeoxyuridine (CldU). Cells were then harvested, and genomic DNA was extracted, combed, and analyzed as previously described (Lebofsky et al, 2006 (link); Herrick & Bensimon, 2009 (link)). The primary antibody for fluorescence detection of IdU was mouse anti-BrdU (Becton Dickinson), and the secondary antibody was goat anti-mouse Alexa Fluor 488 (Invitrogen). The primary antibody for fluorescence detection of CldU was rat anti-CldU (Novus Biologicals). The secondary antibody was goat anti-rat Alexa Fluor 594 (Invitrogen). The length of the replication signals and the distances between origins were measured in micrometers and converted to kilo bases according to a constant and sequence-independent stretching factor (1 μm = 2 Kb), as previously reported (Herrick & Bensimon, 2009 (link)).
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