I tried western blot targeting TTF-1 (Thyroid transcription factor-1) also known as Nkx2.1.
I tested two primary antibodies, one is Invitrogen, Nkx2.1 Monoclonal Antibody (8G7G3/1) and the other is Cell signaling, Thyroid Transcription Factor 1 (TTF-1) (D2E8) Rabbit mAb #12373.
Blocking was performed for up to 3 hours, and the blocking buffer was tested with 5% and 10% BSA diltued by TBS-T.
The primary antibody concentration was 1:500, 1:1000 and 1:2000 O/N (16h) at 4 °C, and the secondary antibody was treated at RT for 2 hours at a concentration of up to 1:10000.
(I have also tried incubation by diluting the primary antibody in 5% BSA)
And all washing steps were performed three times for 10 minutes each.
However, non-specific bands were still detected. In this attempt, we plan to proceed with blocking O/N at 4 °C.
There is no problem with the actin band, but a non-specific band is detected only for TTF-1, so I am asking a question.
Are there any additional ways?
You could further dilute the secondary antibody.
Something to consider: how does the molecular weight of your unspecific band relate to the expected molecular weight? Sometimes it helps to increase the reducing power (beta-mercapto, dithiothreitol) in your SDS loading buffer to decrease unspecific signals.
What about the concentration of the gel? or the type of HPR revelator? I've recently used west Atto (Thermo) for small protein detection and also the time of incubation ( 3 to 5 min) it's also relevant. About the secondary antibody I use 1:3000
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