ch 3 - Proteome and protein purification Doris E. Terry...

Info iconThis preview shows pages 1–13. Sign up to view the full content.

View Full Document Right Arrow Icon
Proteome and protein purification Doris E. Terry Office: Suite 131 Dyson Building terry_doris@yahoo.com
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Proteomics Imaging Proteomics research tools
Background image of page 2
h Types of sample c Biological Plasma Urine Bile Spinal fluid Milk Tissue Cell c Environmental water soil Air sludge
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
h Objectives of sample preparation c Fractionation c Isolation c Concentration/enrichment of analyte c Solubilization of analyte c Purification Removal of particulates/contaminants Desalting c Preservation of analyte
Background image of page 4
h Methodology c Ultrafiltration c Differential centrifugation c Gel filtration c Liquid-liquid extraction (LLE) c Liquid-solid extraction c Solid-phase extraction (SPE) c Immunoaffinity c Supercritical fluid extraction (SFE) c Micro-dialysis c Electroextraction c Protein precipitation c Gel-based and gel-free electrophoresis
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Differential centrifugation
Background image of page 6
h Salting out c Mechanism Most proteins are less soluble at high salt concentrations Salt concentration varies from one protein to another – e.g. 0.8 M ammonium sulfate precipitates fibrinogen albumin c Advantages Simple c Disadvantages Needs additional step to remove the salt
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Dialysis
Background image of page 8
h Ultrafiltration c Mechanism Selective retention or passage of analyte based on molecular weight (MW) Membrane filters available from 3 K – 1,000 K MW cutoff Low MW compounds in the filtrate High MW compounds on the filter c Advantages Simple and can be automated Fast Often sufficient cleanup for mass spectrometry analysis c Disadvantages Does not concentrate sample Filters have finite capacity before plugging Limited sample cleanup
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
,000 Da MW cutoff 100,000 Da MW cutoff Steps for ultrafiltration 1. Select filter 2. Dilute sample 3. Transfer mixture to ultrafiltration tube and centifuge for 15 -30 min at 4,000 g 3,000 Da MW cutoff Dual sample stage filter for multiresidue sample preparation Filtrate
Background image of page 10
h Liquid-liquid extraction c Mechanism Selection of immiscible solvents and conditions c Advantages Many choices of solvents, pH and ion pairing additives to perform the extraction Widely used Concentrates the analyte c Disadvantages Off-line process Often leads to large volume of solvent usage and disposal Sometimes difficult to obtain high recoveries Time consuming
Background image of page 11

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
h Solid-phase extraction (SPE) c Mechanism Analyte of interest eluted from solid support while interferences are retained Interferences eluted while analyte is bound to solid support and subsequently eluted c Advantages any oices f lid pports Many choices of solid supports Quick and easy to automate Minimize solvent waste c Disadvantages Usually an offline step Requires conditioning Dependent on loading and wash solvent, sample volume loaded and mass loading
Background image of page 12
Image of page 13
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 44

ch 3 - Proteome and protein purification Doris E. Terry...

This preview shows document pages 1 - 13. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online