1. 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

2. 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

3. Describe and explain the observation with:
Oil
Food Coloring
Glass slide
Wax paper

4. Students perform protein purification
Protein Purification Movie Clip from Teachers domain
Students perform protein purification

5. Lab 7: Purification of the RFP protein from the other bacterial proteins.
Principles of protein purification:

6. 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.

7. 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.

8. 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.

9. 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

10. 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

11. Bruce Wallace
RFP

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