Size Exclusion Chromatography Stan HitomiCoordinator Math & ScienceSan Ramon Valley Unified School DistrictDanville, CAKirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High School and Delta College, Tracy, CASherri Andrews, Ph.D.Curriculum and Training SpecialistBio-Rad LaboratoriesEssy Levy, M.Sc.Curriculum and Training SpecialistBio-Rad LaboratoriesSize ExclusionChromatographyInstructorsWhy TeachSize Exclusion Chromatography?Powerful teaching toolLaboratory extensionsReal-world connectionsLink to careers and industryStandards basedSize Exclusion ChromatographyKit AdvantagesStandards BasedCan be used in Biology, Chemistry, or Physical ScienceSufficient materials for 8 student work stationsEasy preparationEasy visualization of separationCan be completed in one 45 minute lab sessionStudy how the structure and biochemical properties of molecules are related to their separationWorkshop Time Line8/28/2024IntroductionComparison of different types of column chromatographySeparation of a mixture of biomolecules by size exclusion chromatographyTypes of Column ChromatographyAffinity Hydrophobic Interaction (HIC)Ion ExchangeAnionCationGel Filtration or Size Exclusion (SEC)8/28/2024Affinity ChromatographyUses an affinity tag- allows molecules to bind to the column- specific to the tagged protein of interest - Examples: HIS-Tag, antibody, GST-TagProteins not bound pass through the column A buffer is used to elute the protein from the column 8/28/2024http:/tainano.com/Molecular%20Biology%20Glossary.files/image047.gifIon Exchange ChromatographyBeads in the column are charged Anion - positively (+) charged beads Cation- negatively (-) charged beadsMolecule to be purified will have the opposite charge from the beads in the columnMolecules not binding to the beads pass through the columnA counter-charged buffer is used to elute the molecule of interesthttp:/tainano.com/Molecular%20Biology%20Glossary.files/image047.gifHydrophobic Interaction ChromatographyHICBeads in the column are hydrophobicColumn is treated with a high salt bufferHydrophobic proteins bind to the beadsA lower salt buffer elutes less hydrophobic proteinsA no salt buffer elutes the protein of interestSize Exclusion ChromatographyBeads in column have tiny poresThe mixture of molecules is added to the columnLarge molecules move through the column quickly traveling around the beadsSmaller molecules move through the pores of the beads and take longer to pass through the columnhttp:/tainano.com/Molecular%20Biology%20Glossary.files/image047.gifPrinciples of Size Exclusion Chromatography The mass of beads in the column is called the column bedBeads trap or sieve and filter molecules based on sizeThe separation of molecules is called fractionationSize of pores in beads determines the exclusion limit (what goes through the beads and what goes around the beads)Molecules are dissolved in a bufferPrinciples of Size Exclusion ChromatographySize Exclusion ChromatographyProceduresOverviewWorkstations Student WorkstationItems Number Collection Tubes 12Columns 1Column end-caps 1Pipet 1Lab Marker 1Test tube rack 1 Common WorkstationHemoglobin/Vitamin B mixtureColumn BufferLaboratoryQuick GuideLabel 10 collection tubes sequentiallyLabel last 2 tubes “waste” and “column buffer”Aliquot 4ml of Column buffer into the tube labeled column bufferStep 1:Label collection tubesStep 2:Column BufferStep 3:Prepare the Column Remove the cap and snap off the end of the sizing column Allow all of the buffer to drain into the waste tube Cap the end of the columnStep 4:Add the protein mix to the column Place column in tube 1 Add 1 drop of protein mixStep 5:Add column buffer and collect fractionsCarefully add 250ml of column buffer to the top of the column (2x) and begin to collect drops into tube 1 - Size separation will work best when the column is left undisturbedCarefully add 3ml of column buffer to the columnTransfer column to tube 2 and begin fraction collectionCollect 5 drops of buffer into tube 2 and transfer the column to tube 3Repeat the same collection procedure collecting 5 drops into each tubeCollect 10 drops at tube 10Molecules of interest: Hemoglobin and Vitamin B12Hemoglobin is brown and has a molecular weight of 65,000 daltonsVitamin B12 is pink and has a mass of 1,350 daltonsThe exclusion limit of the beads is 60,000 daltons: Hemoglobin will exit the column first, then Vitamin B12Hemoglobin (Hb) http:/www.pdb.orgwww.nhlbi.nih.gov Normal Cell Sickle CellDNA: CCT GAG GAG CCT GTG GAG Protein: Glu ValMetalloproteinTransports oxygen to the body Found in the red blood cells (RBC)Heme group contains an iron atom which is responsible oxygen bindingSickle Cell Anemia rises from a point mutationVitamin B12 http:/history.nih.govImportant for normal functioning of the brain and nervous systemInvolved in the metabolism of every cell in the body fatty acid synthesis and energy production DNA synthesis and regulationCyanocobalamin Cobalt (Co) central metal ionSynthesized in bacteriaCoenzymeMUT: (Methylmalonyl-CoA mutase) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA, a key molecule of the TCS CycleMTR: methyl transfer enzyme (5-Methyltetrahydrofolate-homocysteine methyltransferase) catalyzes the conversion homocysteine into methionine, an essential amino acid 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