The attached is a γH2AX western blotting result.293T cells were treated with etoposide or DMSO(control),then cells were lysed in RIPA buffer with sonication.0.2 nitrocelluose membrane was used for transfer.Membrane was washed 4-5 times(5min for each time) with TBST(with 0.05% tween).Four replicates were carried out ofr both groups.There are problems with it,one is the high background(which will be worse after longer exposure time),another one is signal in 4# of control is significantly higher than lanes in the control with similar level in etoposide-treated groups.
I have been working on the γH2AX western blotting for a long time,and these problems appears again and again.So I wonder whether someone can give me some advice.
Dear Tang,
regarding your first problem with the high Background you can try to improve your blocking of the Membrane, so the background can be slightly diminished by that. Most antibody producer recommend a way of blocking or you try Protein-free blocking too avoid false right signals. Since you are experienced in yH2AX WB's you can see which one is the right signal.
For your second question I kindly ask if you synchronize your cells prior to treatment since you use cytostatics and yH2AX is as well phoshorylated in S-phase cells so maybe you see signal from replication stress?
Best regards,
Nicole
Dear Nicole,
Thanks very much for your kind answer.
Both 5% nonfat milk and BSA were used for blocking(1h,RT),and they gave similar results.The specifc band comes from γH2AX.
I did not synchronize 293FT cells prior to etoposide treatment,indeed,the observed γH2AX may comes from replication stress.As similar results(stronger signal in the middle lanes) were observed in other samples(8-10w mice brains),so it is more likely that they come from problems with WB.
Another question,should the SDS included in the transfer buffer for γH2AX ?
Thanks.
Best,
Xiaoqiang
Dear Xiaoqiang,
1. Blotto or dry milk is not recommended for blocking when you are probing a Phospho modifications on proteins so please use 5% BSA for blocking.
2. For normalizing H2Ax use H2A antibody.
3. Use 0.2 um pore size PVDF mambrane and play around with transfer conditions. I have used 35 min at 1 mili Ampere current per cm square.
4. SDS is to be added in transfer buffer.
5. etoposide will arrest cells in G1-S phase but I don't think that cell synchronization is needed in this case or not.
Dear Shakyawar,
Thanks for your kind answer.
1.BSA will be used for blocking next time.
2.H2AX was used for control,but not shown here.
3.I have optimized the transfer conditions with semi-dry.
Thanks again.
Best,
Xiaoqiang
- Badges
- Science method