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In Topic: No WB bands for H2AX at all...!
04 September 2012 - 06:15 PM
well pals..! thanks for all ur suggestions as finaly i detected some BANDS..!!!
......hurray
......hurray
In Topic: No WB bands for H2AX at all...!
20 August 2012 - 05:16 PM
OK..! today i am going to start my WB again... following your suggestions.... wish me luck 
In Topic: No WB bands for H2AX at all...!
14 August 2012 - 05:35 PM
ascacioc, on 14 August 2012 - 01:36 AM, 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
Andreea
lsek, on 14 August 2012 - 02:10 AM, 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.
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.
For cell lysis, I use to add some enzyme inhibitors in my lysis buffer (i.e. PMSF: Serine protease and Thiol protease inhibitor. Aprotinin:Trypsin and Chymotrypsin inhibitor. Leupeptin: Trypsin, Plasmin and Papain inhibitor). Now as i am working with a phospho-protein (H2AX), do I need to add phosphatase inhibitors as well? If yes which inhibitors i will need?
