Anti-c-Fos (phospho T325) antibody (ab27793)

The order no was XXX and it was placed on 22.03.12.

ANSWER:

Thank you for contacting us.

I am sorry that this antibody did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

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I have tried the suggestions that you made but am still not satisfied with the antibody. I would appreciate it if you could arrange a refund of this product.

ANSWER:

Thank you for your reply.


I am sorry to hear that my suggestions did not improve your results with this antibody.I am happy to arrange for a refund. Would you please provide your order number for this product?


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

I recently purchased the antibody ab27793 - phospho c-Fos T325 but it has not worked at all well. I have tried it on both mouse embryonic fibroblast and motor neuron lysates. Both cell types had been stimulated with growth factors and so there should have been plenty of phosphorylated protein to detect. I blocked the membranes for 1 hour in PBS-T 0.2% with 5% non-fat milk. I then incubated with the primary antibody (1:1,000) over night at 4 C in PBS-T 0.2% with the addition of 5% BSA. The membrane was incubated with an HRP conjugated rabbit secondary antibody, in PBS-T 0.2% and 5% non-fat milk for 45 min. In between the primary and secondary antibodies and also post secondary antibody the membranes were washes in PBS-T 0.2% for 15 min x1 followed by 5 min x3, although I did try longer washing steps also. There are multiple bands on western blots, some of which vary between membranes. There was also a lot of background signal detected making it very difficult to distinguish what was going on.
Any advice would be appreciated and/or a refund arranged.

ANSWER:

Thank you for contacting Abcam regarding ab27793.


I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the protocol information you provided and would like to make some suggestions to improve your results.


When using a phospho-specific antibody it is highly advised against using milk as a blocking reagent or diluent. I would recommend switching to 5% BSA in PBST instead. Also switching to TBST often decreases background. I would suggest the following:


How much sample are you loading? I would recommend loading 50-75ug total protein in each lane. Then, block for at least 1 hr at room temperature in 5% BSA in TBST. Incubate with the primary antibody (also diluted in 5% BSA in TBST) for 1 hour at room temperature rather than overnight. Test 1:1000, 1:2000, and 1:4000. Perform washes with TBST. Incubate the secondary antibody diluted in 5% BSA (dilute according to manufacturers instructions). It may be necessary to titrate the secondary antibody concentration as well. Wash and develop as you normally would.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or if these suggestions do not improve your results with this antibody and I will be happy to provide a replacement or refund per our Abpromise guarantee.

I notice that you have phospho c-fos antibodies available. Have these antibodies been used for immunofluorescence in tissues?

ANSWER:

Thank you for your enquiry. Ab27793 has not yet been tested for application in IF (only Western blotting so far)but ab17933 - Rabbit polyclonal to c-Fos (phospho T232) - has been tested in IF. There is an image on the online datasheet with more more information.

Please contact us again if you have any additional questions.