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Laboratory : Experiment 2: SDS PAGE Lecture : SDS Polyacrylamide Gel Electrophoresis

Laboratory : Experiment 2: SDS PAGE Lecture : SDS Polyacrylamide Gel Electrophoresis In-Class Writing : review proposals (pages xxxiii-xxxvi & 129-133) Read : Unit 2 (34-35) & JBC 266: 3811-14 (1991) Hand In : proposal (3 copies) Due Next Class : flow chart 4 (day 1; DNA extract).

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Laboratory : Experiment 2: SDS PAGE Lecture : SDS Polyacrylamide Gel Electrophoresis

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  1. Laboratory: Experiment 2: SDS PAGE Lecture: SDS Polyacrylamide Gel Electrophoresis In-ClassWriting: review proposals (pages xxxiii-xxxvi & 129-133) Read: Unit 2 (34-35) & JBC 266: 3811-14 (1991) HandIn: proposal (3 copies) DueNext Class: flow chart 4 (day 1; DNA extract)

  2. SDS- Polyacrylamide Gel Electrophoresis 2-mercaptoethanol: reduces S-S bonds SDS: denatures proteins & imparts uniform negative charge SDS-PAGE separates most proteins by size. Some proteins migrate faster or slower than expected.

  3. stacking gel = 4 % acrylamide, pH 6.8 resolving gel = 15 % acrylamide, pH 8.8 +

  4. His-tagged FnbA protein: 138 amino acids MW = 15,341

  5. MRGSHHHHHHGSGGQNSGNQSFEEDTEEDKPKYGQGGNIVDIDFDSVPQIHGQNKGNQSFEEDTEKDKPKYEHGGNIIDIDFDSVPHIHGFNKHITEIIEEDTNKDKPSTQFGGHNSVDFEEDTLPKVSSGLQPSLISstopMRGSHHHHHHGSGGQNSGNQSFEEDTEEDKPKYGQGGNIVDIDFDSVPQIHGQNKGNQSFEEDTEKDKPKYEHGGNIIDIDFDSVPHIHGFNKHITEIIEEDTNKDKPSTQFGGHNSVDFEEDTLPKVSSGLQPSLISstop M = start; HHHHHH = 6 His tag RGS…GSG; SGL…LIS = vector GQN… KVS = FnbA

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