Protein Characterization Techniques PDF

PROTEIN CHARACTERIZATION AGENDA Purification Identification Sequence 3D Structure PROTEIN PURIFICATION METHODS THE BASIS OF CHROMATOGRAPHY Find something unique to your protein Use it to separate it from the others TBL TYPES OF CHROMATOGRAPHY 1. Gel filtration 2. Ion Exchange...

PROTEIN CHARACTERIZATION AGENDA Purification Identification Sequence 3D Structure PROTEIN PURIFICATION METHODS THE BASIS OF CHROMATOGRAPHY Find something unique to your protein Use it to separate it from the others TBL TYPES OF CHROMATOGRAPHY 1. Gel filtration 2. Ion Exchange In groups of 3-4, work on the 3. Affinity first page of the worksheet. 5 minutes SIZE EXCLUSION CHROMATOGRAPHY  Separating molecules based on size  Use to separate multiple proteins  Use to change or remove salts from solution  Desalting  Big things come out first  Protein size (kDa) 0.1 1 10 100 1,000 10,000 100,000 FRACTIONATION RANGE An SEC column comes with a range of proteins it can separate Anything too small elutes late and broadly Anything too big flows off the column as one band together GEL FILTRATION Practice Question: 1. You pass a purified protein through a gel filtration column and see a peak at 60 kDa. 2. You instead perform the filtration in the presence of 6 M urea you now see a 30 kDa protein peak. 1. What does the urea do? 3. You then filter the protein in the presence of 6 M urea and 10 mM BME, you find a 15 kDa species. 1. What does the BME do? Describe the structure of this protein. FILTERING BY SIZE  Tubes with filters separate based on size  Sample goes on top  Centrifuge to force the solution through  Molecular weight cut off (MWCO)  Based on pore size of filter  > MWCO: Stays up top  < MWCO: Flows through to bottom BASICS OF BINDING Equilibrate  Bring your column to the correct environment Equilibrate Bind  Use 20 column volumes to make sure your whole column is clean and in the right buffer Wash  Column Volume (CV): Based on how much liquid your Elute column holds BASICS OF BINDING Binding Equilibrate  Add your protein solution to the column Bind  Let your protein bind while others flow through Wash  Always collect the flow through just in case your Elute protein doesn’t bind BASICS OF BINDING Washing Equilibrate  Not everything that doesn’t bind will come off Bind immediately Wash  Wash with at least 10 CV to remove everything you can Elute  Collect your wash just in case  Elution BASICS OF BINDING  Get your protein off the column  Done by competing with your protein for binding to Equilibrate the column Bind  Step: Switch directly from wash buffer to elution Wash buffer Elute  Gradient: Slowly change to the elution buffer  Good if somethings don’t come off in the wash ION EXCHANGE CHROMATOGRAPHY (IEC)  Beads have a charge opposite to those on your protein  Choose a buffer that maximizes your charge  Positive: Acidic (amines positive, carboxylics neutral)  Negative: Basic (amines neutral, carboxylics negative) ION EXCHANGE CHROMATOGRAPHY (IEC)  Elution Options  Increase salt (compete for charges)  Change pH (adjust charge of your protein)  What could you add to your protein to increase the affinity? AFFINITY CHROMATOGRAPHY The beads are specially designed for interactions with your protein Proteins are specially designed to include a “tag” that will interact with the column of choice IMAC immobilized metal affinity chromatography NICKEL COLUMNS  Proteins engineered with multiple histidines at one end  6xHis but can go up to 10xHis  What would more histidine do?  How do you get the protein off the column? Imidazole AFFINITY CHROMATOGRAPHY Nickel columns are the most Column Tag Elution common (cheap, easy, reusable) Ni-NTA Histidine Imidazole There are a lot of options out Glutathione GST Protein Glutathione (reduced) there for affinity Amylose Maltose Binding Protein Maltose Antibody FLAG-tag Denature or FLAG Nanobody SPOT-tag SPOT or pH Streptavidin Strep-tag Desthiobiotin Albumin BSA pH, heat, or urea Calmodulin CPB EGTA Halo-link HaloTag Protease Amylose Protein G Low pH IDENTIFICATION SDS-PAGE SDS: Sodium Dodecyl Sulfate PAGE: Polyacrylamide Gel Electrophoresis Separates proteins based on size Analytical Technique A small amount to know if you were successful WHAT’S IN A GEL?  SDS: Denatures, adds charge  Bigger proteins = more charge  Polyacrylamide: Creates a mesh that the proteins have to travel through, separates by size  BME: Reducing agent Rice University has a “Hall of Shame” for SDS-PAGE TBL SDS-PAGE Complete pages 2-4 on the worksheet YIELD vs PURITY Yield  How much protein did you make/retain on this step? Purity  How many contaminants are in the sample? ISOELECTRIC FOCUSING (IEF) The isoelectric point (pI) of a protein is the pH at which the protein carries a net neutral charge Based on 1. The sequence of your protein 2. The charged residues accessible to the outside SEPARATING BY CHARGE  Proteins are put in a gel with a pH gradient  When the current flows, they move towards the cathode or anode  They stop moving when they no longer have charge at that pH You can do this with denatured protein or in their native state! COMBINING TECHNIQUES Your research mentor hands you a mixture of four proteins. PRACTICE YOUR SCIENCE!  Propose a method for the isolation of Protein B from the others. Protein pI MW (kDa)  Propose a method for the isolation of A 4.1 80 Protein A from the others. B 9.0 84 C 8.8 27  If Protein A carried several sequential Histidine residues (that bind nickel) its D 3.9 172 N-terminus, how would you revise your method? SEQUENCING MASS SPECTROMETRY More commonly called Mass Spec Proteins are digested and the pieces assembled to know the sequence Trypsin cuts after Arg and Lys Chymotrypsin cuts after aromatics (Trp, Tyr, Phe) PROTEIN MASS SPEC LC/MS  LC/MS: Liquid Chromatography and Mass Spec  Liquid chromatography uses the hydrophobicity of the fragments to separate them  As each fragment elutes, mass spec is used to determine the size of the peptide LC: Elutes at different times MS: Each peak has multiple pieces at different sizes 3D PROTEIN STRUCTURE 3D STRUCTURE  X-Ray Crystallography Technology improves the way we image proteins in their three-  NMR dimensional structures  CryoEM  Computational X-RAY CRYSTALLOGRAPHY  The most used method and the “oldest”  Entire field dedicated to making crystals  Some even require space missions! HOW DOES IT REALLY WORK? OPTIONS FOR MAKING X-RAYS Synchrotron Linear Accelerator  Multiple users at once  A lot bigger!  Slightly more compact  Only one user at a time  A few around the world  Steady stream of particles  Fast acceleration Stanford Linear Accelerator 2 miles long NMR Can determine structure, but better for interactions Good for proteins that are a little more flexible Still requires a lot of computational work CRYO-EM  “Frozen” is an understatement  Samples are flash frozen in liquid oxygen (