I have recently been carrying out western blots on Arabidopsis thaliana leaf samples.
The gene of interest is overexpressed using a 35S promoter and is tagged to an HA epitope (with no spacer region between).
I am unable to detect any HA protein in my samples. To confirm the function of the Anti-HA primary and secondary antibodies, I used a positive control of a purified HA protein. This did show up on my Western Blots. However this protein was not a protein produced in planta. The antibodies used have apparently worked previously in in tobacco.
Also, I am able to detect the overexpression of the gene of interest using RT-qPCR so it should be present in my samples.
Has anybody got any suggestions as to why I am unable to detect HA in my sample?
Thank you for your help