Part 4 - Video Lecture Notes
Sunday, February 10, 2019 1:27 PM
Purification Analysis
Reason To purify a protein To check if sample is
Protein Sequencing (identifying the sequence) pure
What happens after Continue w/ purification Discard sample that was
technique? analyzed
• Step #1: Cut up proteins into smaller peptides using proteases. Researcher can mon
How much sample is Entire sample is used As small a portion as protein purification: #
used? possible. bands decrease after
○ Sequence each peptide cut by a protease, sequence peptides cut by another protease.
Examples Column Chromatography PAGE (electrophoresis) fractionation step.
Precipitation: Salt Fractionation, pH SDS-PAGE
○ Traditional method is to sequence one protein at a time using Edman's degradation. change 2D PAGE?
Antibody-based: Western Blots,
Immunoprecipitation
○ Modern method is to sequence multiple proteins at a time using Mass Spectrometry. (Proteomics)
• Identify amino acids of each peptide fragment.
○ Edman's degradation: Remove each N-terminus a.a. from one end IN ORDER.
○ Mass spec: sequencing multiple peptides RANDOMLY.
• Once sequence of each fragment is known, determine order of fragments:
○ Cut same protein w/ different proteases.
○ Sequence both set of peptides.
○ Find first peptide of one set of peptides. Find the corresponding peptide in the other set.
○ Find the next peptide in the first set by determining which peptide is overlapping with the other set.
○ Continue with all the fragments-> peptides should have the same a.a. sequence and order as original
protein!
Sunday, February 10, 2019 1:27 PM
Purification Analysis
Reason To purify a protein To check if sample is
Protein Sequencing (identifying the sequence) pure
What happens after Continue w/ purification Discard sample that was
technique? analyzed
• Step #1: Cut up proteins into smaller peptides using proteases. Researcher can mon
How much sample is Entire sample is used As small a portion as protein purification: #
used? possible. bands decrease after
○ Sequence each peptide cut by a protease, sequence peptides cut by another protease.
Examples Column Chromatography PAGE (electrophoresis) fractionation step.
Precipitation: Salt Fractionation, pH SDS-PAGE
○ Traditional method is to sequence one protein at a time using Edman's degradation. change 2D PAGE?
Antibody-based: Western Blots,
Immunoprecipitation
○ Modern method is to sequence multiple proteins at a time using Mass Spectrometry. (Proteomics)
• Identify amino acids of each peptide fragment.
○ Edman's degradation: Remove each N-terminus a.a. from one end IN ORDER.
○ Mass spec: sequencing multiple peptides RANDOMLY.
• Once sequence of each fragment is known, determine order of fragments:
○ Cut same protein w/ different proteases.
○ Sequence both set of peptides.
○ Find first peptide of one set of peptides. Find the corresponding peptide in the other set.
○ Find the next peptide in the first set by determining which peptide is overlapping with the other set.
○ Continue with all the fragments-> peptides should have the same a.a. sequence and order as original
protein!
