Question (54128) | Anti-HPV16 E6 + HPV18 E6 antibody [C1P5] (ab70)

Go to datasheet (ab70)

Question


About your questions and suggestions for ab70 antibody, the customer
answers are below:
She did the assay again, changing her protocol as suggest, however the
unspecific staining remains.
answers:
1- Her cells are monolayer, and she fixed with PFA for 20 min
2- The staining is correct, and it is strong enough at negative
control. She did a control using only secundary antibody (FITC)- w/o
primary antibody, and it did not stain. See attached ppt.
3- Yes, she reduced the incubation time of primary antibody (2h),
and even so the negative control remained stained
Please, see attached ppt.
Best regards,

Answer

Thank you for your reply.

As we have not previously tested this antibody in ICC/IF, we could not guarantee that it would work for this application, as under our Abpromise we only guarantee it to work in WB, IP& IHC-P.

However, I think that the customer may be seeing some specific staining using the antibody, as although there is a signal in the negative control (without HPV or BPV), the signal in the positive cells is much more intense. Although it is difficult to make a accurate comparison, as the images were taken at different magnification.

To help reduce the background, that is being seen in the negative control, I would suggest the following:

1 - Increase blocking to 10%BSA, for 1hr at room temperature.

2 - Reduce the primary antibody concentration to 1/750, for 2 hrs at room temperature.

Hopefully, the above protocol variations will help to improve the staining that is being seen.

If there is anything else I can help you with, please let me know.

注册