Quantifying the interaction ofSodium Dodecyl Sulfate andMytilus californianus footprotein 1Department: Molecular, Cellular,and Developmental Biology (MCDB)Intern: Lauren RehbeinMentor: Dusty Rose MillerAdvisor: Herbert Waite
Goal One: mcfp-1 Extraction and PurificationRemove Foot Fillet Epithelium HomogenizeVariousChemical Steps
Goal One: mcfp-1 Extraction and PurificationSize Exclusion ColumnChromatography; 40 µl at0.6ml/min. on ShodexKW802.5.Gel Electrophoresis(Acid Urea Gel)Absorbance (280nm)Elution Profile:5 10 15 20 25 30Minutes10 11 12 13 14 15 16 17 18 19
Fuchsin Dye Assay: TheoryProblem: No direct way to quantify SDS in solutionSolution: React SDS with fuchsin dye and measureabsorbance at 553nmSDS-Aqueous SolutiondyeH HN+H 3 CNH 2H 2 NAqueous SolutionIon pairNH 2chloroformOO S OOchloroform-+H HNH 3 CH 2 N
Basic Fuchsin AssayStandard Curve of SDS:R 2 =0.96x=y/0.0132 +50.92
At what proportion does SDS interact with mcfp-1?Basic Fuchsin Assay:SDS concentration (nM)10080604020SDS PAGE:04/10% GelMcfp-1 appears to be interactingLESS with SDS than a typical protein.Mcfp-1 does NOT precipitate SDS!
Conclusion• SDS does not precipitate mcfp-1.• Mcfp-1 may be interacting less with SDS than a typicalprotein!§ Artifact of detection method?§ Fuchsin assay may only be applicable to protein precipitationinteractions.Original observation was in M. edulis• This highlights yet another difference in mfp-1 of M. edulisand M. californianus.Further Direction: Repeat experiment with M. edulis mefp-1.
ReflectionsI learned from this experience that:• Proving something is difficult.• Lab research is extensive.• Relationships are important to establish.• Maintaining a balanced life, both work and play, is essential!I enjoyed my time in MCDB, and I am considering majoring inscientific studies.
AcknowledgementsThank you to all who helped me during my fourweeks of research, presentations, and fun. Your helpis truly appreciated.v Dusty Millerv Materials Research Dpt.v Professor Herbert Waitev MCDBv Lubi Lenaburgv Internsv Chris Hammetterv Professor Alison Butlerv Julia Callenderv CNSIv CSEPv LEAPS