BIOL 200 Lecture 6: Purification, Detection and Characterization of Proteins

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Purifying Proteins

• Separates proteins based on different properties

• We can obtain proteins from cells

Protein separation through chromatography

• place soluble protein sample in a liquid phase inside column

• Sample will move through solid phase, different proteins move at different speeds

Gel Filtration Chromatography

• separating proteins based on their size

1. Beads in solid phase will have small pores that interact with sample.

2. Large proteins will not fit through pores and will fall straight to the bottom

3. smaller proteins stuck in pores are then washed out and collected

Ion exchange chromatography

• separating proteins based on charge

1. Beads (solid medium) are given a charge

2. Proteins with same charge will fall through, while proteins with opposite charge will bind

3. Proteins are then washed with solution alike to them in charge. weaker bonded proteins fall sooner because they are outcompeted for the bonds more easily.

Antibody affinity chromatography

• Purify proteins based on antibody interactions

1. Any proteins that don’t bind to the antibodies on the beads fall through

2. Proteins that did bind (had correct antibodies) are then removed by lowering the pH to weaken the binding.

Electrophoresis

1. protein mixtures loaded in wells

2. voltage applied so proteins migrate from negative to positive side

3. Smaller proteins move through the pores faster

4. Direction of migration is determined by net charge (attracted or repelled to +)

5. Speed of migration determined by mass/charge ratio

What does SDS do in gel electrophoresis?

• denatures proteins

• hydrophobic tail of SDS interacts with hydrophobic amino acids inside protein.

• gives all proteins uniform negative charge.This allows proteins to be separated based solely on their size during electrophoresis.

What is SDS - PAGE?

• A type of electrophoresis used to study protein that infects phages

• All proteins look the same, just different lengths

Immunoblot

• Used to detect specific proteins in an SDS - PAGE sample

• sample transferred to membrane that exposes protein surface

• primary antibody detects an exposed protein

• tagged secondary antbody binds to primary antibody (fluorescent or radioactive)

Co-Immunoprecipitation

• Antibodies that only recognize certain proteins

• used to purify other proteins in a complex that interact with it

• Samples used are not treated, maintaining tertiary and quarternary structures

• Immunoprecipitate contains protein which carries a structure that antibodies recognize (epitope) and partner proteins

• Antibody recognizes GR which interacts with a second ligand and PPARa

Immunofluorescence

• antibodies used to locate proteins in cells and tissues

1. Cell put in solvent that ensures structure preservation

2. Primary antibodies detect proteins

3. Tagged secondary antibodies attach to primary antibodies, allowing visualization under a fluorescence microscope.

Fluorescence

1. Electrons named fluorophores absorb photons, causing electrons to get excited and move to a higher orbital

2. when they return to ground state they emit light

3. excitation-emission cycles are oxidized to stop producing fluorescence.

How does a fluorescent microscope work?

1. Uses a filter to block and direct light

2. Light hits filter and reaches dichroic mirror that reflects green light and lets red light through

3. We view green fluorescence through objective lens under the microscope.

4. Red light is passed through the emission filter.

Gene fusion

• done by cloning, it is used to mark proteins of interest

• Endogeneous fusion involves homologous recombination. C or N terminal is chosen depending on which end the protein is.

• All resulting proteins will be marked by fluorescent protein.

• Ex. GFP keeps cells alive to allow for real time tracking of proteins we are interested in.Gene fusion is a technique where two genes are joined together, often used to attach a reporter gene, like GFP, to a target protein, enabling visualization and tracking within living cells.