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