Immunoprecipitation/Endocytosis

Immunoprecipitation

Purification of a specific protein using antibody binding

1. Suitable antibody is added

2. Antibody binds to protein of interest

3. Protein A or G added to make antibody protein complexes insoluble

4. Centrifugation of solution pellets antibody-protein complex. Removal of supernatant and washing

Co-Immunoprecipitation

Purification of a specific protein using antibody and detecting binding partner in IP

Controls:

1. Both X and Y are present in whole cell lysate

2. IP should have X, then look for Y (did we actually purify X? If not, we know Y won't be there)

3. Do IP with random antibody to see how precipitation of X or Y

Late Endosome

After the early endisome. When pH is low, it fuses with the lysosome and everything inside is degraded by the lysosome

Endocytosis

The process by which cells take in fluid, as well as large and small molecules, by invagination of the plasma membrane

Phagocytosis

Involves ingestions of large particles (even other cells or organisms) into large vesicles (usually >250 nm), usually by specialized cells (cellular eating)

Generally used as a defense mechanism and most commonly found in immune cells.

Actin helps form the pseudopods that wrap around the desired import.

Phagosomes

The large vesicles brought in that fuse with lysosomes

Pinocytosis

Involves ingestions of fluid and small molecules into small vesicles (cellular drinking)

For immune surveillance and nutrient uptake. Almost all cell types do this.

Receptor-Mediated endocytosis

Similar to pinocytosis, but involves specifically enriching which molecules are taken up

Cholesterol Intake

Typically done through Receptor-Mediated endocytosis.

Many viruses bind to cholesterol receptors to hijack and get into the cells.

LDL binds to LDL receptors and is endocytosed. The vesicle fuses with the endosome, which has a low pH, so cargo is released.

The receptors are recycled back to the membrane.

The endosome fuses with the lysosome. The lysosome destroys the LDL and releases cholesterol.

LDL

A cholesterol carrier

Endogyneous cholesterol

Cholesterol made in our body

Acetyl CoA can be converted into cholesterol and used. This is not done if we are already bringing in cholesterol from diet

Exogenous cholesterol

Cholesterol that comes from outside the cell

Autophagy

When damaged/non functioning organelles are broken down by the lysosomes.

Familial hypercholesterolemia

Led to accumulation of LDL, causing heart disease in young people.

When exogenous cholesterol was added to cells, the endogenous cholesterol synthesis did not stop. However, it did stop when the LDL was added in a vesicle.

The mutation was on the receptor, preventing LDL from getting into the cells properly.

HMGcoA

An enzyme required for endogenous acquisition of cholesterol (cholesterol synthesis)