I am trying to detect any differences in DNA damage/apoptosis related proteins by western blot between wild type and disease cells. I ran an initial WB and saw no bands for either protein and am now looking to optimise this, however I have seen signal in both cell types with the same antibodies via immunofluorescence. I don't know if I didn't see protein signal because of the WB protocol (ie gel type, run and transfer voltages and times etc). These proteins are ~17kDa each, antibodies: http://www.merckmillipore.com/GB/en/product/Anti-phospho-Histone-H2A.X-Ser139-Antibody-clone-JBW301,MM_NF-05-636#anchor_REF and http://www.merckmillipore.com/GB/en/product/Anti-Caspase-3-Antibody-active-cleaved-form,MM_NF-AB3623. I used 4-15% tris-glycine gels, 100V 30 mins run, 200V 1 hour transfer as this was my standard WB protocol for a 30kDa protein, I understand this probably needs optimising for a smaller molecular weight but I thought I would start with a standard protocol.
I have been looking into creating a positive control to detect these proteins. Some things I have come across include 0.5uM staurosporin or UV treated HeLa. Is it possible to irradiate the HeLa in a standard laminar flow hood as this is my only source of UV light? If yes, any suggestions on how long to leave the cells in the hood (without lid plastic lid and media for maximal exposure)?
Thank you!
It's a good idea to have "positive control" conditions with the cells you are using.
For a damage condition, I rather recommend DNA damaging chemicals because it is much easier to control than UV light in the hood which you have not calibrated.
Phleomycin from InvivoGen, for example, is not too expensive. Our student detected very clear increase of gamma-H2AX after 2 hr treatment with 100 µg/ml Phleomycin in several cell lines.
Shin-Ichiro Hiraga Thank you for your advice, I will look into Phleomycin. Could you tell me how the gamma H2AX was detected? Did you use WB and if yes could you let me know gel types, %, run and transfer conditions please?
Hi,
Here are the answers to your questions:
- We use Cell Signaling Technology #9718 gamma H2AX antibody, but I believe your antibody is also good.
- Yes, we are doing western blot
- Either 4-15% or 4-20% TGX gels from Bio-Rad are used, usually Stain-Free
- We use Bio-Rad Turbo Transfer (semi-dry with proprietary transfer buffer) system with "mixed MW" condition
- We use H2A.X antibody (Abcam ab11175) as a loading control.
I hope this helps.
Shin-Ichiro Hiraga Thanks for the details, I already used the same 4-15% BioRad gel so at least I can cross that off the list of potential problems with my protocol. I'm not aware of the transfer system you mention, I currently use a wet transfer tank from BioRad. I'll keep troubleshooting.
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