Protein Localization

In vivo

Inside a live cell

In vitro

Inside a test tube

Immunofluoresence

Tagging proteins with fluorescence of different colors. When done In vivo, it mostly just tells you what structures, but not where exactly

Cell fractionation

Bursting open cells and separating the organelles to analyze the proteins In vitro

1. Preparing cell extracts: 4 standard methods. A) high frequency sound B) use of a mild detergent C) Force cells through a small hole with high pressure D) Shear cells between a close fitting rotating plunger and the thick walls of a glass vessel

2. Centrifugation - separates by size and density (can be ultracentrifuge). Different speeds will separate differently

3. Confirm if fractionation worked and detect if protein of interest is present, potentially using a microscope or gel electrophoresis

Whole Cell Lysate/Extract

The membrane-enclosed organelles concentrated outside of a cell

Supernatant

The smaller and less dense components following centrifugation

Pellet

The larger, more dense components after centrifugation

Centrifuge Speed

1000x gravity for 10 minutes - Pellet 1 is whole cells, nuclei, and cytoskeletons

20000x gravity for 20 minutes - Pellet 2 contains mitochondria, lysosomes, and peroxisomes

80000x gravity for 60 minutes - Pellet 3 containers closed fragments of ER and other small vesicles

150000x gravity for 180 minutes - Pellet 4 contains ribosomes, viruses, and large macromolecules

Immunoblottong/Western Blotting

1. Separation - Proteins are separated by electrophoresis and a detergent

2. Transfer - Transfer from gel to a membrane

3. Staining - Stain the membrane using antibodies

4. Visualization - appears as a dark color

Blotting Step 1: Separation

Sample is treated with SDS (a detergent), which denatures the protein to a single negatively charged polypeptide. If the protein has subunits, they will separate.

They are then ran through gel electrophoresis and travel towards the positive anodes, with smaller proteins moving faster. They are compared against a ladder

Blotting Step 2: Transfer

The girls are transferred using the sandwich method, essentially sopping up into sponges, which then travel to various places

Blotting Step 3: Staining

Treat the membrane with antibodies that are labeled and will bind to the desired molecule. It is then treated with a second antibody, that recognizes the first antibody and binds. Usually the second antibody has a color indicator to show where it's bound.

The primary antibodies are usually called alphaA or antiA