Western Blot Primary Antibody

This step takes place after the membrane has been blocked with 5% BSA in TBST for around 30 or 45 minutes.
For half membrane size, like the one in the video, use 2 "squares" of parafilm to encapsulate the membrane. Along with that, only 500 ul of solution (5% BSA in TBST) and 1 ul of primary antibody is needed for a membrane of this size.
In the rare scenarios where you are using 1 primary antibody on the whole membrane, use 1 ml of solution with 2 ul of primary antibody and use 3 "squares" of parafilm to encapsulate it.
Tips and Clarification:
- Always check the molecular weight of the primary antibody you are using to make sure you are using the correct section of the membrane.
- Always check what secondary antibody to use and write it down on the tape that it is on top of the washing container.
- Be aware of the membrane's orientation at all times so that you can ensure the membrane is placed face down onto the primary antibody that is on top of the parafilm.
- When you are trying to get the air pockets out between the parafilm and the membrane, only the face down side matters to have no air pockets. It is okay if the top side has air bubbles as the proteins aren't on that side.
- You will lose some of the primary antibody solution during the "flattening." All that matters is that the contact on the front facing side of the membrane is covered in a thin layer of the solution.