Immunodetection will be performed in Lab 11. Blotting modules can be left overni

Question

Immunodetection will be performed in Lab 11. Blotting modules can be left overnight in their tanks at room temperature or sandwiches can be dismantled and the membranes can be placed in blocker at 4 degree C overnight. If blots need to be stored for longer periods, they can be stored submerged in blotting buffer or wash buffer at 4 degree C for up to week. Why are proteins blotted from the polyacrylamide gel to a membrane? Why is it for the gel to be in complete contact with the membrane without any air bubbles? Why do proteins migrate from the gel to the membrane? Can you think of a way to determine if the transfer of a stained gel has been successful? An unstained gel?

Explanation / Answer

1. The proteins are transferred out of the gel and blotted into the membrane in order to facilitate the antibody detection of myosin.

2. It is adviced to remove any type of air bubbles because they prevent proteins from being transferred.

3. The electric current forces the negatively charged proteins to migrate towards the positively charged end of the membrane side.

4. Stained gel: Proteins will be stained blue on the membrane and will be seen on the gel.

Unstained gel:  Prestained protein standards will be visible on the membrane

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