No WB bands for H2AX at all...! - (Aug/14/2012 )
Running the gel at wrong voltage (100V) and time (2.5hrs)?
Using wrong PVDF membrane size (0.2uM )?
Transferring proteins at wrong current (200) or for wrong time (1h and 15min)
Using wrong dilution of primary antibody (1:500)?
Using wrong dilution of secondary antibody (1:10000)?
What kind of transfer are you using: semidry or wet? It might be the case that yur protein is too small and is blotted through the membrane while actin is large enough to remain on the membrane. Add a second membrane on top of your membrane to check whether your protein is transfered through the first one. Do the antibody detection for both of them. Do you activate your PVDF with methanol before preparing the transfer sandwich?
Andreea
Hi kokoakash,
Size of the histone protein is very small. You can try to transfer the protein at shorter time, e.g. 10 to 20 min which will be more than sufficient. Some people double layer the PVDF as precaution.
One very important note, histones are quite basic (positively charged). Although residual SDS should be able to neutralize this and render it negatively charged during SDS-PAGE, the amount of SDS is not sufficient/diluted during the transfer step and hence reverting the histones back to positively charged. If that is the case, you will sometimes see a faint histone band or not at all. To circumvent this problem, reverse the position of the PVDF during transfer, or you can wedge the SDS-PAGE gel with PDVF at both sides of the gel for this purpose (one for capturing the histone at the cathode while the other is to trap the actin protein at the anode).
Useful reference: http://www.millipore.com/immunodetection/id3/proteintransfer
Good luck.
ascacioc on Tue Aug 14 09:36:32 2012 said:
What kind of transfer are you using: semidry or wet? It might be the case that yur protein is too small and is blotted through the membrane while actin is large enough to remain on the membrane. Add a second membrane on top of your membrane to check whether your protein is transfered through the first one. Do the antibody detection for both of them. Do you activate your PVDF with methanol before preparing the transfer sandwich?
Andreea
lsek on Tue Aug 14 10:10:54 2012 said:
Hi kokoakash,
Size of the histone protein is very small. You can try to transfer the protein at shorter time, e.g. 10 to 20 min which will be more than sufficient. Some people double layer the PVDF as precaution.
One very important note, histones are quite basic (positively charged). Although residual SDS should be able to neutralize this and render it negatively charged during SDS-PAGE, the amount of SDS is not sufficient/diluted during the transfer step and hence reverting the histones back to positively charged. If that is the case, you will sometimes see a faint histone band or not at all. To circumvent this problem, reverse the position of the PVDF during transfer, or you can wedge the SDS-PAGE gel with PDVF at both sides of the gel for this purpose (one for capturing the histone at the cathode while the other is to trap the actin protein at the anode).
Useful reference: http://www.millipore...proteintransfer
Good luck.
Yes you should use phosphatase inhibitors. Sodium orthovanadate (Na3VO4) is one of the most commons (I think), not sure of concentration but I know that some commercially available buffers have it at 1mM.
Also, try to block with other agents (milk has phospho proteins in it and can affect your detection).
Do you have a positive control to confirm your antibody is working? Have you titrated your antibody?
well pals..! thanks for all ur suggestions as finaly i detected some BANDS..!!!
......hurray
me likey 
and happy for you
Thanks Andreea..!
Koko,
Glad you overcame problem of no bands for H2AX antibody. Could you please post here what are the changes you made to make it work for you?
Thanks,
-Cyp19ab
kokoakash on Wed Sep 5 02:15:34 2012 said:
well pals..! thanks for all ur suggestions as finaly i detected some BANDS..!!!
......hurray