Proteomics

RNA editing

a molecular process through which the sequence of an RNA molecule is altered post-transcriptionally, leading to variations in the protein encoded by the corresponding gene.

alternative splicing

portions of the mRNA can be either introns or exons depending on the action of spliceosomes which can change based on environmental conditions.

mRNA Degradation

mRNA can be actively degraded or protected from degradationto change the amount of protein produced.

Post-translational Modification Types (5):

Proteolytic cleavage, protein degradation, protein-protein interactions, glycosylation, and phosphorylation.

Post-transcriptional Modifications (3):

RNA editing, alternative splicing, and mRNA synthesis and degradation.

myofibrils

basic contractile element of muscle cells, bundled in muscle fibers.

Sarcomere

Linear series of contractile units within each myofibril

5 things SDS-PAGE can tell you about proteins

Molecular weight, purity, quantity, number of proteins, and differences between protein samples.

Laemmli sample buffer contains:

Tris buffer, SDS, Bromophenol blue, and glycerol.

What is a common source of error in protein identification using PAGE?

Flashcard #3
A common source of error is the migration of different proteins similarly, making it difficult to distinguish between them.

Reducing agents with a strong smell

DTT and BME, which break disulfide bonds and reduce background bands.

Factors for protein denaturation:

Heat, ionic detergent (SDS), and reducing agent

Precision Plus Protein kaleidoscope standard

Used for creating standard curves to estimate unknown protein sizes and estimating unknown molecular masses.

APS and TEMED

An initiator/catalyst is needed to cast a gel that polymerizes acrylamide and provides cross-linking for the gel structure.

Discontinuous System

Stacking gel (top) and separating/resolving gel (bottom). It allows for better resolution of protein bands by concentrating the sample before it enters the resolving gel.

The purpose of SDS

override protein charges, allowing mass to be migration factor, and maintain linear form by denaturation of proteins.

BME

Beta mercaptoethonol

DTT

dithiothretiol

Actin and myosin standard purpose:

Positive control that should be present across all samples examined.

If actin and myosin standard did NOT show up,

there must be an issue in electrophoresis,sample loading, or detection.

Why is Coomassie used?

To visualize bands using bromophenol blue added.

Western Blotting

Technique used to identify a protein by transferring proteins from a gel to a membrane and probing with specific antibodies.

Primary structure

Denatured linear chain of amino acids

Secondary structure

Domains of repeating structures, such as pleated sheets and alpha helices

Tertiary structure

3D shape of folded polypeptide, maintained by disulfide bonds, electrostatic interactions, and hydrophobic effects.

Quaternary structure

Several polypeptide chains associated together to form a functional protein.