Protein Extraction Flashcards
What are the forms of protein extraction?
Sonication, French press, osmotic shock, digestion, detergents, homogenization
What is sonication?
metal probe vibrates at high frequencies which creates waves through the test tube; this breaks apart the cells; vibrations create heat which may denature enzymes; may also require single cells because vibrations not penetrate tissues
What is french press?
pressure; a threaded piston spins and pushes down the liquid, creating pressure in the solution; the pressure will then be released very quickly, causing the cells to burst open; this is good for eukaryotic cells when you want to keep the nuclei and organelles in tact
What is an anion exchange column?
Where the stationary phase is + and the target compounds are negative; they will interact more strongly with the stationary phase and elute in the later volumes
What is a cation exchange column?
Where the stationary phase is -, and the target compounds are positive; they will interact more strongly with the stationary phase and elute in the later volumes
What is osmotic shock?
Cells are introduced into a hypo-osmotic solution, which causes them to swell and burst open, releasing their contents; works best on cells without cell walls
What is digestion
Enzymes digest the cells
What are detergents?
Break down the cell membrane to release the cell’s contents
What is homogenization?
Using force to break things apart, such as a blender; necessary if we have multicellular samples, such as tissues
What is activity?
How many Units of enzyme per mL (concentration)
What is specific activity?
The amount of Units of enzyme per mg protein
How is yield calculated?
total Activity before purification/total activity before purification; ideally we want it to stay the same but in reality we will lose some of our targeted protein
How is purity calculated?
S.A. after/S.A before; we want this to go up because it means we have more of our target protein per all protein in the sample
Order of specificity for chromatography
Affinity (most, but most expensive), Ion exchange, size exclusion (least, easiest to do)
What are the ingredients to make the 5x buffer for SDS-Page?
10% SDS, 20% glycerol, 0.2M Tris HCl (pH 6.8); 0.05% bromophenol blue; 10mM beta-mercaptoethanol
Briefly describe the SDS-Page procedure
Protein samples are boiled for 5 min in 5x loading buffer; Gel is cast and poured between glass or plastic plates; combs are used to form loading wells; gel is placed vertically in electrophoresis apparatus where top and bottom tanks are filled with buffer; samples and ladder are loaded into the wells, apparatus is turned on to 250V and runs till blue dye reaches the bottom; once finished, gel is removed and immersed in: coomassie blue for 10 min, then buffer for 10 min (repeat 2-3x); OR silver nitrate for more sensitive applications (,pre complicated and time consuming) OR fluorescent dye staining (sensitive, but requires specialized equipment)
what purpose does glycerol serve in the buffer?
to increase density and viscosity so protein can sink to the bottom and separate
the dye must be the same charge as the SDS (negative) so it runs in the same direction
ye
i think coomassie blue is the least sensitive protein staining method we studied but idk
ye
What is the purpose of a stacking gel?
To help increase band resolution
What composes a stacking gel?
The top gel (stacking gel) made up of 5% acrylamide, pH 6.8 with Tris-HCl; Bottom gel (running gel) with ~12% acrylamide, pH 8.8 w/ Tris HCl; upper buffer: pH 8.3 w glycine, pI=6
Why would some parts of standard curve not be linear?
The proteins travelling may be too big or too small to be effectively separated by the acrylamide gel at that specific concentration
What is the horizontal plane of a 2D protein gel?
size (also called dimension 2)
What is the vertical plane of a 2D protein gel?
charge (also called dimension 1)
