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
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
Describe and explain the observation with:OilFood ColoringGlass slideWax paper
Protein Purification Movie Clip from Teachers domain
Students perform protein purification
Lab 7: Purification of the RFP protein from the other bacterial proteins.
Principles of protein purification:
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.
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.
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.
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)
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
RFP
Bruce Wallace
Purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer
Bruce Wallace