separating cells

FACS - fluorescent antibody-prelabeled cells

chromatography
	- charge (ion exchange)
	- size (gel filtration)
	- binding (affinity)
	- polarity (hydrophobicity)
	
	
cell characteristics

cytosolic conditions are reducing, meaning that things inside a cell want to grab hold of electrons. the opposite is true for the extracellular face of cells, and inside of organelles like mitochondria, peroxisomes, lysosomes, etc

cells maintain varied concentrations of ions:
low sodium
high potassium
very low calcium
hydrogen conditions yielding neutral pH (7.2)

membrane transport

lipid membranes allow free diffusion of water and nonpolar molecules, but polar molecules need to be transported as well. the class of membrane transport proteins tackle this problem. These proteins get the job done either requiring no energy in the case of facilitated diffusion, or with energy for active transport. The electrochemical gradient across a membrane is responsible for the existence of these two forms of transport. Transporters are actually subdivided into the carrier proteins, which undergo conformational changes in order to transport a molecule across the membrane, and channel proteins, which form aqueous pores through which smaller solutes can travel. 

example: sodium-potassium pump. An ATPase antiporter carrier protein which uses one ATP to export three sodium ions in exchange for two potassium ions. This pump is electrogenic because its actions create negative ionic conditions intracellularly. The pump also affects the osmolarity of a cell; if the pump operates for too long, a solute concentration will develop extracellularly, causing water to leave the cell. 

Channels: 

Not all molecules can move through channel proteins unchecked. Many channels are actually gated, which is triggered either by a change in voltage, mechanical force, or ligand binding event. Ligands may be signal transmitters, ions, or nucleotides. 

The most common channel is the potassium leak channel, which allows slow extracellular flow of potassium.


Protein sorting:
signal sequences, cleaved off by peptidases, eg, nuclear import signals, ER localization
signal patches, 3d truly biometric conformations - membrane transport through pores
glycosylations - proper protein folding (chaperone recruitment), stability in solution, protection from degredation
lipid modification - localization to membranes
ubiquitination - targeting to the proteasome complex or localization using SUMO


lysosome vs peroxisome:
lysosome = garbage disposal
perosisome = toxin disposal

lysosome buds from the trans golgi network, peroxisome is self replicating
both are acidic, oxidating environments, but peroxisomes get rid of toxins (formaldehyde, hydrogen peroxide, phenol) using oxidative enzymes (oxidase, catalase) and require a lot of oxygen, whereas lysosomes use such oxidase and hydrolases to digest protein, nucleotides, lipids, phosphates, etc. both use ATP to pump H+ into the organelle.

transgenic animals - random integration of recombinant DNA:
1 - transforming ES cells
	- select an ES cell which expresses gene of interest
	- inject into blasocyst of embryo
	- implant into mouse (in vitro fertilization)
	- grown up mouse is a chimera
	- hope the ES cell made it to germ line
2 - transfection of gene into pronucleus of fertilized egg
	- transfect vector into pronucleus of egg, hopefully homologous recombination
	- implant egg into uterus
targeted animals - site specific integration of DNA
	- makes mosaic mice
	- cre lox system