{[ promptMessage ]}

Bookmark it

{[ promptMessage ]}

L6-8 - Berg Tymoczko Stryer Biochemistry Sixth Edition...

Info icon This preview shows pages 1–8. Sign up to view the full content.

View Full Document Right Arrow Icon
1 Biochemistry Sixth Edition Chapter 3: Exploring Proteins and Proteomes Berg • Tymoczko • Stryer Most biological fluids are a complex mixture of many proteins ( e.g. milk) •MALDI-TOF can be used to determine MW of proteins •Chpt 3: We’ll focus mostly on 3.1 (introduction to protein purification), 3.5 (mass spec), 3.6 (NMR and X-Ray crystallography), with a brief discussion of the other sections. •Genome =DNA sequence of an organism •static in size •Proteome =Proteins expressed by the organism •size varies w/ development, cell type, ... •much larger than number of genes
Image of page 1

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

View Full Document Right Arrow Icon
2 Section 3.1: Protein purification: Need an assay to track purity •Assay example: NADH production •Goal: Maximize specific activity=rate/[protein] total •Done by removing other proteins from sample absorbs at 340 nm no absorption at 340 nm Differential centrifugation allows different cell fractions to be assayed Salting out allows simple purification and concentration of proteins •Different ammonium sulfate (0.1 to 5 M) ‘cuts’ causes different proteins to precipitate •Then need to desalt sample: Dialysis water goes into bag MWCO=1-100kD commercially available stir bar
Image of page 2
3 Gel filtration chromatography Biggest proteins elute first your protein elutes! Ion-exchange chromatography •elute with NaCl gradient •your protein of interest: net positive •beads: overall negative your protein elutes! Cation exchanger Anion exchanger or ‘Sephadex’
Image of page 3

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

View Full Document Right Arrow Icon
4 Affinity Chromatography beads; have G your protein of interest; binds G your protein elutes! HPLC (or FPLC) leads to excellent resolution of complex protein mixtures •‘hplc’ h igh p ressure (p erformance) l iquid c hromatography •‘fplc’ f ast p rotein l iquid c hromatography •resin is more finely divided=better separations! Polyacrylamide gel electrophoresis (PAGE) smaller proteins larger proteins Pipette Buffer to power supply
Image of page 4
5 PAGE crosslinking ( 29 : 1 is typical ) •a crosslink •makes pores in gel •more crosslink=smaller pores SDS has two jobs for PAGE: 1.) Charges up the protein in a (somewhat) non-specific way 2.) Denatures the protein Example of a PAGE gel of proteins--Coomassie-blue stained Top of gel Bottom of gel M ‘M’=marker lane low MW protein high MW protein
Image of page 5

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

View Full Document Right Arrow Icon
6 PAGE mobility has a semi-logarithmic dependence on MW •unknown protein •mobility of 0.46 MW of 29kD Isoelectric focusing acidic protein (=low pI) basic protein (=high pI) gel has a pH gradient due to presence of ampholytes •pI is the pH at which charge on protein =0 •When pH=pI, protein does not electrophorese positive electrode (Early in run) (End of run) 2-D electrophoresis 1st dimension of electrophoresis (=IEF) 2nd dimension of electrophoresis Band has 5 proteins Band has 3 proteins 1 2 3 1 2 3 4 5
Image of page 6
7 A real 2-dimensional gel a protein of interest •For E. coli •>1000 proteins resolved Purification of a protein: step-by-step large decrease Small decrease large increase Small decrease large increase
Image of page 7

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

View Full Document Right Arrow Icon
Image of page 8
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