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No WB bands for H2AX at all...! - (Aug/13/2012 )

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Hi,
I am a PhD scholar working with HL-60 cells and I am trying to induce DNA damage to them and detect gamma H2AX proteins (15kDa) by western blotting using a well-documented toxin, Arsenic.
However, instead of seeing a band at the expected 15kDA region, I've detected nothing after repeating the experiment several times. However, my actin band appeared whenever I detected for H2AX and actin simultaneously but surprisingly, there were no band at all for H2AX...! Only once i got some light and merged bands at 15kDa but at that time no actin band appeared..!
I am very sure that the H2AX proteins are in the cells because I am using high molar concentration of arsenic that is sufficient to induce DNA damage.

where i am going wrong:
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)?

Here is my protocol:
1. Lyse the cells. centrifuge at top speed for 30mins at 4deg Celsius to remove cell debris.
2. Quantitate amount of proteins and load 20ul of protein sample onto a 13% gel. Run gel at 100V for 2.5hrs.
3. Transfer proteins to a 0.2uM PVDF membrane at1h and 15min.
4. Block membrane in freshly prepared 5% milk (in TTBS) at room temp for 1 hr.
5. Incubate membrane with anti-phospho-histone H2AX, diluted 1:500, overnight at 4deg Celsius.
6. Wash with TTBS (3X). Incubate with Rabbit HRP conjugated IgG (diluted 1:10000) for 1hr at room temp.
7. Wash with TTBS (3X) and detect with Immobilon Western Chemiluminescent HRP Substrate.

Please help me out with this problem of mine..
I shall be very thankful to you.

-kokoakash-

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

-ascacioc-

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.

-lsek-

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.
Thanks, well I use wet transfering. I am greatly thankful to you for your kind suggestion.
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?

-kokoakash-

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?

-almost a doctor-

OK..! today i am going to start my WB again... following your suggestions.... wish me luck

-kokoakash-

well pals..! thanks for all ur suggestions as finaly i detected some BANDS..!!! ......hurray

-kokoakash-

me likey :) and happy for you

-ascacioc-

Thanks Andreea..!

-kokoakash-

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

-Cyp19ab-
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