What are the best antibodies to use as markers of cell cycle stages?
Cyclin A and CDC2 are expressed in the G1/S transition; During S phase E2F/DP family; Late S phase CDC25C and cyclins B1 and B2 are upregualted; Cyclin B2 is involved in regulating G2/M progression of cell cycle. Hope this helps.
Are you staining for microscopy or flow cytometry? If the latter, I would recommend a BrdU assay.
Here is the best paper for you...
http://www.cyto.purdue.edu/cdroms/cyto10a/cytometryhistory/individualhistories/media/darz/cyclins.pdf
Kind regards,
Steve
I think this depends on the technique you would like to employ. In immunohistochemistry, antibodies specific for the Ki-67 antigen will label all cycling cells (G1 to M). To mark mitotic figures, use an antibody against pHH3. I am not aware of any antibodies which will label cells specifically according to cell cycle phase. If you use viable cells, BrdU assay will help to identify cells in S-phase.
Seeing that you are working in Birmingham, talk to Paul Murray. Give him my regards.
Running a BrdU assay will allow you to identify all of the cell cycle phases in your cell population. We run the assay in combination with PI. Trying different time points will help to indicate the proliferation of your cell type. If you want to have specific markers, you could detect the cyclins specific for the different phases of the cell cycle by western blot, and correlate that with your BrdU data.
As suggested before, looking at BrdU-incorporating cells by cytometry in parallel with PI or 7AAD staining is a good way to follow your cells through the cell cycle.
Using EdU (instead of BrdU) increases the signal-to-noise ratio as it uses a small molecule (fluorophore-coupled azide) instead of an antibody, and you don't need to denature the DNA.
Thank you for your replies. I am setting up a BrdU-incorporation assay for Flow Cytometry. However I also want to try Western blotting of key cell cycle proteins and was wondering if anyone had any success at this and if so what proteins they specifically looked for?
Cyclin D1 from Thermo Scientific (RB-010-P) is a great antibody. Our lab got an aliquot of cyclin A from Santa Cruz (sc-751), but you had to use the primary at 1:200. I would check several journal papers and see how the antibodies for the different cyclins look like and then proceed from there.
I have found this list of anti-BrdU antibodies.
http://goo.gl/DUXY9n
Hope that's helpful!
Hi thanks for you reply. I am mostly interested in showing that my cells are in S phase, but as I have had issues with my block I think that my cells may be getting stuck in a mitotic state so I want to show whether or not this is the case.
The best antibodies for flow are from BD. You can do flow for Cyclins as well as EdU in a multi- parameter experiment providing you have multiple lasers. Include a Hydroxyurea, Mimosine, double Thymidine or Aphidicolin block in addition to Nocodazole.
Hello, My cells are arrested in S phase and I am doing EDu labelling assay. I am keeping Hydroxyurea as a positive control for DNA damage. If I am doing Edu, Do I still need to do Brdu assay? I am trying to do western blot of Cyclins A, D E, B. These suggestions are really very helpful! Please let me know your views. Thanks a lot!
Avani
No you do not, since both assays basically work the same by incorporating BrdU and EdU respectively into the DNA during the replication. In addition to Cyclin's you might also want to use other markers such as phosphorylated RB.
Hi, Can I take advantage of your post and ask what would you recommend if the goal was to identify the cell cycle phase between G0-M by immunofluorescence?
Thanks for any suggestion
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