Prin cell bio cell separation

cell separation- used to collect cells of interest

FACS, Velocity sedimentation, selective surfaces, others

Cell fractionation-separates to see the cell organelle of interest

Differential cen-trirugation, rate zonal-equilibrium-density gradient (all mean the same thing)

Protein purification/enrichment

ion exchange chromatography, gel filtration, affinity chromatography, SDS gel electrophoresis, Notir, 2-D gel electrophoresis, others

Tissue dissociation

uses EGTA which is a calcium chelation→calcium dependent cell adhesion molecules, protase- cleaves proteins: EX. Trypsin, colleagues, liberase 

FACS- Fluorescene activated cell sorting

Type of flow cytometry, machine

Guava-flow cytometer

doesnt separate and sort cells, counts cells based on fluorescence, multicellular plate

Velocity sedimentation

no fluorescence dyes required, separates cells based on speed through gravitational field

Selective surfaces

purify the hematopoietic erythropoietin receptor

EX. Dynabeads

Veridexs CellSearch system

Counts CTC(circulating tumor cells), mass binds to CTC, mass have iron, has magnets

Cell fractionation

used in Lyse cells

• sonication-ultra sound

  • Destroys all cell membrane

• Detergent- soluble cell membrane

• Multiple freezing cells

• Trituration

  • Uses very small bore needles

  • Mechanical sheer, causes cells to lyse

  • Organelles are ok

Differential centrifugation

lyse cells

Have to be in buffer, making sure there’s no major shift in pH because of lysosomes- pH 5.0

• addition of protease inhibitors- inhibit lysosomal enzymes

Michrosomes

rough microsomes

• have ribosomes attached to them

• Resealed rough endoplasmic reticulum

Smooth microsomes

• resealed outer cell membranes

Equilibrium density centrifugation

separates entities based on their abilities to reach an equilibrium density- the density of the medium

Protein separation techniques

Column chromatography (liquid chromatography)

• ion exchange chromatography

  • Preserves active function 

  • Charged beads

  • Non-ion covalent interaction with beads and proteins

  • Nail is used to elude proteins from the beads based on their attraction 

• Gel filtration

  • based on physical size

liquid chromatography

column

Preserve the native structure and function

Affinity chromatography

Beads can bind a protein to it

Native gel electrophoresis

requires gel, acrylamide gel acts like a sieve

Native structure and function is preserved

SDS gel electrophoresis

Requires acrylamide

Denatures proteins

Isoelectric focusing

isolates proteins based on isoelectric point

Point in a pH filed with no more movement

2-D gel electrophoresis

best of the 4 terms of resolution

Distinguish a phosphorylation protein from its non-phosphorus parent

Western blots

identify a protein of interest using gel electrophoresis

• virtues

  • Qualitative- identifies the protein of interest and see molecular weight

  • Quantitative- change in abundance of protein of interest

western blots and photo affinity amino acids

no UV light of some part and some UV used for the protein of interest

SELDI-TOF/MALDI-TOF

identifies proteins based on molecular weight

Resolution is the best- one amino acid

PARTS

• laser

• Top detector

• Sample chip

Alzheimer’s disease

amyloid B-42

Protein A immunoprecipitation

protein A binds to most Fc ends of mAbs

Autoradiography

use of radioactive tracers to follow a cellular event

Approach

• CHO cells

• Add to culture

• In cells PO4→ATP

• Add insulin

• SDS gel electrophoresis

• Autoradiology