Anti-Cleaved PARP antibody (ab2317)

Thank you so much for your prompt reply. In response to your question, I am staining dissected pupal epidermal samples in order to image dendritic arborization neurons using confocal microscopy. The ab2317 antibody has been successfully used before in other labs for this very purpose, and has been documented in several publications. Below is a detailed protocol that I have been following for the sample preparation and protocol.


Staining Procedure:

7. Immediately fix samples in 4% PFA diluted in 1X PBS for 30 min at room temperature (I have increased the fixation time to at most 1 hr during troubleshooting).

8. Rinse fixed pupae 3x, 5 minutes each in 1X PBT-X (1X PBS + 0.3% Triton-X-100).

9.Block samples in 1X PBT-X + 5% normal goat serum for 1 hr at 4 degrees Celsius.

10. Incubate with 10 ug/mL rabbit-anti-cleaved PARP diluted in 5% normal goat serum/PBT-X overnight at 4 degrees Celsius.

11. The next day, remove antibody dilution and wash samples 3x, 10 minutes each in 1X PBT-X at room temperature.

12. Incubate in secondary antibody 1:200 goat-anti-rabbit-Alexa 488 diluted in 5% normal goat serum/PBT-X for 2 hrs at room temperature.(also have tried Alexa 568 and Cy5-conjugated secondary antibodies)

13. Wash samples 3X, 10 minutes each in 1X PBT-X.

14. Equilibrate samples in glycerol-based mounting media for at least 30 minutes.

15. Separate epidermis from pupal case and mount on cover slip and slide. Store samples at -20 degrees.

I am also co-staining these samples with a mouse-anti-GFP antibody-this staining has been constantly strong and clean which suggests that my sample preparation and staining procedure is working. I'd appreciate any help on this matter. I have been troubleshooting this staining for months and it would be great to have some advice. One other thing I was wondering is if anyone has had any issues with the particular lot of antibody that I purchased or with this antibody in general.

Thank you again for getting back to me so quickly and for your help. I look forward to hearing from you soon.

ANSWER:

Thank you for your reply with the protocol information.


Based on the protocol information you provided, it seems that you are using relatively standard methods that should be compatible with the antibody. One possibility would be to try a different fixative, such as methanol or acetone. Additionally, blocking (and diluting your antibodies)with 3% BSA rather than serum may also help your staining.


I have checked our records and we have not had other users report any difficulties using this antibody. I am happy to offer a replacement or credit if these suggestions do not improve your results. I look forward to your reply so that I may assist you further.

We purchased an antibody froagainst cleaved PARP (It is the rabbit polyclonal to cleaved PARP ab2317). However, after many attempts for immunofluorescence detection, I have been unable to see any immunoreactivity of this antibody using the concentrations recommended in the data sheet supplied with the antibody (dilution of 1:10-1:20, both dilutions were attempted) and with conditions successfully used in previously published reports. I was wondering if anybody has reported issues with this antibody recently. I am using standard staining techniques and using it in Drosophila pupae expressing a UAS-CD8::PARP-Venus reporter driven by Gal4 expression, as similarly used in Williams et al 2006, Nature Neuroscience and Rumpf et al 2011, Development. Any suggestions/information would be much appreciated. Thank you in advance and I look forward to hearing from you.

ANSWER:

Thank you for contacting Abcam regarding ab2317.


I am sorry that you have been experiencing difficulties with this antibody in your experiments. I was hoping you would provide me with some additional information so that I may assist you in resolving this issue. In particular, are you staining whole pupae, tissue sections, or cells in culture? As the antibody is specifically validated for ICC I want to be sure you are using this application. Would you also send me a detailed summary of your protocol and sample preparation? If possible, I would be happy to offer some protocol recommendations, or alternatively a replacement or credit per our Abpromise guarantee.


I look forward to your reply so that I may help you further. Please do not hesitate to contact us if you have any additional questions.