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: 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: Oil Food Coloring Glass slide Wax paper

Students perform protein purification 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.

After dilution – like equilibration buffer 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: Students 3+4: Spin Lysate Pass 3000 ml Equilibration B through column. Dump liquid. 5 min Spin Load 500 ml RFP Carefully Collect 250 ml Supernatant Into new tube Pass 1000 ml Wash B Add 2000 Elution B. COLLECT RED PROTEIN In TUBE! Add 250 ml Binding Buffer. Vortex. Cleanup (ask teacher) Pass 2000 ml Equilib. B

Last day of labs Lab 7: Check protein by fluorescence. Denature by boiling – what happens? 2) Lab 7 Conclusions 3) Cleanup: Fill tips, Pipettes, racks 4) Notebooks: Label all assignments, journals. 5) Reflection – Towards review

Bruce Wallace RFP

Purification of RFP from an overnight culture Bruce Wallace Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer