Lecture 3 - lab techniques (continued)

Western Blot technique (steps)

Used to answer: is a protein there? how much of the protein is there?

1. Lyse (burst open) cells, spilling out all their proteins

2. Sort proteins by size (SDS-PAGE): load proteins into a gel and run electricity through it. proteins are coated with SDS, a detergent that gives them all a negative charge, so they all get pulled down towards the positive electrode at a rate determined ONLY by size.

3. Transfer (blot) proteins into a membrane (like pressing a wet newspaper onto a paper towel to make a copy) → this preserves the proteins’s exact positions

4. Bathe the membrane in a blocking solution so that your antibodies stick ONLY to the target protein

5. Antibody tagging (primary antibody: add an antibody that binds ONLY to your target protein) (secondary antibody: add a second antibody that ONLY binds to antibodies, tagged with something that produces light)

6. Add a substrate that reacts with the tag on the secondary antibody → produces light → expose it to a camera → wherever your protein sat on that gel, you see a dark band

Immunoprecipitation (IP) technique (steps)

Used to investigate quaternary structure and answers the question: “what proteins does my protein interact with?”

1. Lyse cells (burst cells open) to release proteins into a solution

2. Add an antibody “bait” into the solution that specifically binds to your protein of interest

3. Fish antibody out of solution (precipitation), this is due to the antibody being attached to resin beads/magnets

4. Wash the beads to rinse off everything so that you ONLY see your protein of interest and whatever other protein is bound to it

5. Run a western blot for the protein bound to your protein of interest → If you see a band for the pound protein, that means it interacts with your protein of interest

BU-MS technique

Used to answer: “what IS this protein, down to its exact sequence/identity?”

1. Chop the protein into peptide pieces with trypsin

2. separate and weigh those pieces with mass spec

3. shatter them again to read their sequence

4. match the pattern to a database to identify the protein.

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