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 Transcript for the LECTURE video: Introduction to Hydrophobic Column Chromatography Slide 1 (title). Purify RFP by Hydrophobic Column Chromatography by Jo Wu, ABE-­‐Fullerton College Slide 2. Obtaining Red Fluorescent Protein. During the Amgen Biotech labs so far, you have already transformed E coli bacterial cells with the recombinant pARA-­‐R plasmid. When grown on agar plates containing ampicillin and arabinose, these cells produce pink colonies. If these transformed cells are cultured in liquid broth for 24 hours, you will have millions of bacterial cells producing the red fluorescent protein, as seen in this flask. As each bacterial cell produces about several thousand different proteins, how can the RFP be separated and purified from the bacterial proteins? Slide 3. Purification of RFP from an Overnight Culture • We transfer the pink liquid from the liquid culture flask into a centrifuge tube. • After centrifugation, you see two layers. The top liquid is the liquid broth which should be discarded in a 10% bleach waste beaker. All of the bacterials cells are now in the bottom pink pellet. • We now add Lysis buffer to this pink pellet, resuspend the cells, incubate, freeze and thaw two times. This will help ensure that all of the bacterial cells will be lysed open and release all of the proteins. • Centrifuge again, but notice that the top liquid supernatant is now pink and the pellet at the bottom is white. The pellet contains the cell debris. What you want to keep is the pink liquid, which should be a mixture of bacterial proteins and RFP. CLICK. Transfer the pink liquid supernatant into a new microfuge tube and Add binding buffer. • This will be the protein mixture that you will separate by hydrophobic column chromatography, so that you will end up with purified Red fluorescent protein. Slide 4. Column Chromatography Column Chromatography is a general technique to separate a mixture of proteins based on their chemical content. There are many different types of chromatography used in biochemical and biotechnology labs, but we will focus on the hydrophobic interaction chromatography technique to purify RFP. Slide 5. Parts of Chromatography Column Most chromatography columns look like this diagram. There is a column that contains buffer solution, a resin bed, a stock cock valve. It is very important that you do not allow the resin bed to ever run dry. Use the stock cock valve to control the flow rate of the column. Slide 6. Red Fluorescent Protein, RFP All proteins have both hydrophobic and hydrophilic amino acids, but RFP is considered a highly hydrophobic protein overall. This is the reason that hydrophobic interaction chromatography was chosen as the purification technique. Transcript: “Intro to Hydrophobic Column Chromatography”, ABE-­‐FC, August 2015 1 Slide 7. Surface Hydrophobicity Different types of proteins have different 3-­‐D shapes, and also different hydrophobic areas. The bottom 4 diagrams are rough sketches indicating that the blue hydrophobic regions can vary between different protein molecules. Slide 8. HIC Chromatography Concept The large blue circle here represents a Chromatography Resin bead. It has been coated with hydrophobic groups. Under high salt conditions, hydrophobic areas will interact with other hydrophobic areas. So if hydrophobic areas on proteins are exposed, they will bind to the hydrophobic coating on these resin beads. Slide 9. Binding Buffer = High Salt Concentration After the column has been prepped and equilibrated, you will be introducing the protein sample and high salt binding buffer solution. This sample contains numerous bacterial proteins as well as the red fluorescent protein. If there are any proteins that are mainly hydrophilic, they will not bind to the resin beads and pass through the column quickly. But if there are proteins that have exposed hydrophobic areas, these proteins will bind and stay on the resin beads. Slide 10. Wash Buffer = Medium Salt Concentration After the binding buffer has flowed through the column, we will next add a WASH buffer which has a medium salt concentration. If there are any proteins that are moderately hydrophobic, they will be released from the resin beads and flow down the column. That leaves only the most hydrophobic proteins to still be bound to the resin beads. Slide 11. Elution Buffer = Low Salt Concentration We will then add the ELUTION buffer that has very low salt concentration. This condition causes the hydrophobic interactions to be interrupted, so that the hydrophobic proteins are now released from the resin beads and flow down the column. RED FLUORESCENT PROTEIN will be one of these proteins, and be easy to find due to its pink color. Slide 12. RFP Spectroscopy and Fluorescence You will want to collect the pink liquid that comes down the column into a clear microfuge tube, as that should be purified red fluorescent protein. Your class may want to have a contest to see which group obtained the most or the purest RFP. You can check the results by looking at RFP with visible light, with a blue light box or an uV transilluminator. You can also use a spectrophotometer or microplate reader with 500-­‐540 nm excitation and 570-­‐610 emission wavelengths. Slide 13. Lab Technical Tips • Drip liquid slowly down the wall of the column, so that resin bed is not disturbed. • Do NOT allow resin bed to dry out. Stop the liquid by turning the valve to horizontal position, when liquid is just above the resin bed. • When collecting RFP, place a white paper behind column for easier viewing. Slide 14. Credits • This video project has been sponsored by funding by Amgen Biotech Experience and Embitech. • For more information about the ABE program, including additional videos related to the ABE curriculum, please visit: www.amgenbiotechexperience.com and ABE-­‐LA.org. • Power point by Jo Wu, Ph.D. Narration by Jessica Wu-­‐Woods. Video Production by Adam Brown. Transcript: “Intro to Hydrophobic Column Chromatography”, ABE-­‐FC, August 2015 2