liquid cultures to lysis : movie clip from teachers domain
DESCRIPTION
Lab 7 – Purification of RFP protein ( mFP ) from an overnight culture Day 1: Collection of Bacteria and cells lysis (break down). Liquid cultures to Lysis : Movie Clip from Teachers domain. Student Guide Lab 7. Day 1 - Materials needed : Per group : - PowerPoint PPT PresentationTRANSCRIPT
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Liquid cultures to Lysis:Movie Clip from Teachers domain
Lab 7 – Purification of RFP protein (mFP) from an overnight culture
Day 1: Collection of Bacteria and cells lysis (break down)
Student Guide Lab 7
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Day 1 - Materials needed:Per group:-1 test tube with 1.5ml (full!) bacteria(additional 1ml will be added by your teacher). Altogether: 2.5 ml.-250 ul of Elution Buffer (EB)-40 ul of Lysozyme (Lys)- a P-1000 pipetter, Tissue paper.Per class: -Microcentrifuge, Vortex
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Describe and explain the observation with:OilFood ColoringGlass slideWax paper
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Protein Purification Movie Clip from Teachers domain
Students perform protein purification
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Lab 7: Purification of the RFP protein from the other bacterial proteins.
Principles of protein purification:
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1. Proteins differ in their amino acid sequence, and so..2. Proteins differ in how polar or non-polar (hydrophobic they are).3. The more hydrophobic they are, the stronger they will bind a hydrophobic surface.4. We will isolate RFP based on its greater hydrophobicity, compared to the other bacterial proteins.
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5. Chromatography: separation of chemicals according to their different affinity to the surface, as compared to the solvent. 6. Molecule with a greater relative affinity to the solid surface will be more delayed from coming out from the column. 7. The higher the salt concentration, the more attached to the column.
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Solution Salt Effect
Binding 4M After dilution – like
equilibration buffer
Equilibr
-ation
High
2M
Hydrophilic proteins out.
Hydrophobic proteins stay.
Washing Low
1.3M
Less hydrophobic proteins
out, RFP stays.
Elution none RFP out.
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Students 1+2:Spin Lysate
5 min Spin
Carefully Collect250 ml SupernatantInto new tube
Students 3+4:Pass 3000 ml Equilibration Bthrough column. Dump liquid.
Add 250 ml Binding Buffer. Vortex.
Load 500 ml RFP
Pass 1000 ml Wash B
Add 2000 Elution B. COLLECT RED PROTEIN In TUBE!
Pass 2000 ml Equilib. BCleanup (ask teacher)
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Last day of labs1) Lab 7: Check protein by
fluorescence.- Denature by boiling – what
happens?2) Lab 7 Conclusions3) Cleanup: Fill tips, Pipettes, racks4) Notebooks: Label all assignments,
journals.5) Reflection – Towards review
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RFP
Bruce Wallace
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Purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer
Bruce Wallace