I'm actually confused about something. I just started using Western blot and usually refer to my seniors about the protocol. The problem is, one of my seniors only use primary antibody to bind with the protein (N protein). How about secondary antibody?Is that unnecessary?
For my project,I have to detect his-tag connected to the protein to make sure the his-tag is still connected to my protein after harsh cell disruption. Should I use primary antibody only (anti-his antibody) or secondary antibody also? If I should use secondary antibody, can i use anti-mouse to bind with anti-his antibody?
I really appreciate if somebody can explain this.