Recitation__2-Simona

Recitation__2-Simona - 7.05 Section 05 TA: Simona Tescu...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
7.05 – Section 05 02/20/2008 TA: Simona Tescu Email: tescu@mit.edu 1/6 RECITATION #2 Covers lectures: 3, 4, 5 (Protein purification, structure and folding) Reading Assignment (Stryer): Lecture 3 (pages 65-89), Lecture 4 (pages 93-101, 39-50), Lecture 5 (pages 50-59) Recitation #3 will cover lectures: 6, 7, 8 Exam #1 is next Friday, Feb 29 th , covering lectures 1-8! During next week’s recitation you will also receive a package with practice problems for the exam. Solutions to the problems we don’t have time to cover during recitation are available during office hours. Overview of theory + Practice Problems Protein purification Steps in protein purification: - Lyse the cells by grinding, freezing/ thawing, sonication, rapid pressure changes, or by using a detergent - Centrifuge at increasing speeds to get read of nuclei, mitochondria, microsomes (membranes and ribosomes); keep the supernatant - “Salting out” the supernatant: separates the proteins based on solubility; adding (NH 4 ) 2 SO 4 to the solution increases the ionic strength of the solution which decreases the solubility of different proteins at different rates - Dialysis: removes salts of low MW ((NH 4 ) 2 SO 4 ) and other contaminants, using a semipermeable membrane which does not allow proteins to pass - Gel filtration chromatography: separates proteins based on size o Use porous beads made of carbohydrates; small proteins travel slower through the column than larger proteins because small proteins can enter the beads - Ion-exchange chromatography: separates proteins based on their charge o Use anion-exchange columns (DEAE-diethylaminoethyl) to select for negatively charged proteins o Use cation-exchange columns (carboxymethyl) to select for positively charged proteins o Then, use a higher concentration of ions (negative ions for negatively charged proteins and positive ions for positively charged proteins) to elute the protein of interest - Affinity chromatography: separates the proteins based on their affinity to a specific substrate (for example glucose); then elute protein using a higher concentration of the specific substrate (in our case, glucose)
Background image of page 1

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

View Full DocumentRight Arrow Icon
7.05 – Section 05 02/20/2008 TA: Simona Tescu Email: tescu@mit.edu 2/6 P1) Complete the table below: Purification procedure Total protein (mg) Total activity (units) Specific activity (units/mg) Purification level Yield (%) Crude extract 20,000 4,000,000 1 100 (NH 4 ) 2 SO 4 precipitation 5,000 3,000,000 DEAE-cellulose chromatography 1,500 1,000,000 Size-exclusion chromatography 500 750,000 Affinity chromayography 45 675,000 Specific activity of a protein (SA) = Total activity/ total amount of protein P2) Problem Set 1, 2008, Question 2 b: As you purify a protein: - Total activity will…. -
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 02/06/2009 for the course 7 7.05 taught by Professor Unknown during the Spring '09 term at MIT.

Page1 / 6

Recitation__2-Simona - 7.05 Section 05 TA: Simona Tescu...

This preview shows document pages 1 - 3. Sign up to view the full document.

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