# Protein Technologies ## Protein Engineering ### Intentional Mutagenesis > See also: > - [[Antibody Engineering]] ## Protein Quantification There are several standard steps done to - [[Lowry Assay]] - [[Bradford Assay]] ### Important Light Absorbance Values > [!important] Important Light Absorbance Values > > **260 nm** -> DNA and RNA concentration > **280 nm** -> Protein concentration > **340 nm** -> CDNB Assay, NADH (in Pyruvate Kinase/Lactate Dehydrogenase Assay) > **595 nm** -> [[Bradford Assay]] > **600 nm** -> Bacterial growth in LB media > **750 nm** -> [[Lowry Assay]] > ## Protein Purification ![[Pasted image 20240426132732.png|150]] ![[Pasted image 20240426132751.png|150]] ### Chromatography - Size Exclusion Chromatography - [[Chromatography]] ### Acquiring Protein #### Inducing Protein Expression > See also: [[Model Systems]] There are several **protein expression systems** that can be used to express target proteins of interest: **In vitro (cell-free expression system)** - Only requires a ribosome with the mRNA of interest. - Fast, simple, produces pure protein - Expensive, small amounts of protein produced, protein folding may differ in non-cellular environments **Prokaryotic** - Easiest to work with since they readily take up DNA plasmids - Produce the most **Yeast/Plant Cells** - aa **Baculovirus (Insect Cells)** > [!summary] Mammalian Cells > Advantages > - a > Disadvantages > - **Mammalian Cells** - Ensures that the product is in its native state (assuming the target protein is from humans) - ![[Pasted image 20230308150939.png|500]] Bacterial expression systems are by far the easiest of the cell-based systems to work with, as prokaryotic cells easily take up plasmid DNA and grow very quickly. - [[Cloning and Expression Vectors]] ## Protein Concentration & Storage ### Protein Concentration Recall methods to concentrate proteins after purification (lyophilization, ultrafiltration, reverse dialysis, column concentration). d i a l y s i s ### Protein Storage Recall methods to store proteins, and the role of various stabilization compounds such as cryoprotectants (glycerol, ethylene glycol), protease inhibitors (PMSF, EDTA, EGTA), chelating agents (EDTA), reducing agents (DTT, β-ME), and antimicrobial agents (sodium azide). ## Protein Analysis - [[Western Blot]]