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

Info icon This 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 [email protected]
Image of page 1

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

View Full Document Right Arrow Icon
Proteomics Imaging Proteomics research tools
Image of page 2
handptright Types of sample circle6 Biological Plasma Urine Bile Spinal fluid Milk Tissue Cell circle6 Environmental water soil Air sludge
Image of page 3

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

View Full Document Right Arrow Icon
handptright Objectives of sample preparation circle6 Fractionation circle6 Isolation circle6 Concentration/enrichment of analyte circle6 Solubilization of analyte circle6 Purification Removal of particulates/contaminants Desalting circle6 Preservation of analyte
Image of page 4
handptright Methodology circle6 Ultrafiltration circle6 Differential centrifugation circle6 Gel filtration circle6 Liquid-liquid extraction (LLE) circle6 Liquid-solid extraction Solid-phase extraction (SPE) circle6 circle6 Immunoaffinity circle6 Supercritical fluid extraction (SFE) circle6 Micro-dialysis circle6 Electroextraction circle6 Protein precipitation circle6 Gel-based and gel-free electrophoresis
Image of page 5

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

View Full Document Right Arrow Icon
Differential centrifugation
Image of page 6
handptright Salting out circle6 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 (blood clotting protein), & 2.4 M precipitates serum albumin circle6 Advantages Simple circle6 Disadvantages Needs additional step to remove the salt
Image of page 7

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

View Full Document Right Arrow Icon
Dialysis
Image of page 8
handptright Ultrafiltration circle6 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 circle6 Advantages Simple and can be automated Fast Often sufficient cleanup for mass spectrometry analysis circle6 Disadvantages Does not concentrate sample Filters have finite capacity before plugging Limited sample cleanup
Image of page 9

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

View Full Document Right Arrow Icon
3,000 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 Dual sample stage filter for multiresidue sample preparation Filtrate
Image of page 10
handptright Liquid-liquid extraction circle6 Mechanism Selection of immiscible solvents and conditions circle6 Advantages Many choices of solvents, pH and ion pairing additives to perform the extraction Widely used Concentrates the analyte circle6 Disadvantages Off-line process Often leads to large volume of solvent usage and disposal Sometimes difficult to obtain high recoveries Time consuming
Image of page 11

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

View Full Document Right Arrow Icon
handptright Solid-phase extraction (SPE) circle6 Mechanism Analyte of interest eluted from solid support while interferences are retained Interferences eluted while analyte is bound to solid support and subsequently eluted circle6 Advantages Many choices of solid supports Quick and easy to automate Minimize solvent waste circle6 Disadvantages Usually an offline step Requires conditioning Dependent on loading and wash solvent, sample volume loaded and mass loading
Image of page 12
Image of page 13
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern