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  • 1. IdentifyingPOU5F1Duy NguyenBiomedMr. Hyke
  • 2. What is POU5F1?• Another name OCT 4 (octamer-binding transcription factor 4)• POU domain, class 5, transcription factor 1• Transcription factor of the POU protein family• Required during embryo genesis• Molecular weight 38,571 Da
  • 3. Escherichia coli• Bacteria found in the intestine of mammals• Helps the body break down and digest food, produce vitamin K and fight against other pathogens in the intestines• Very cheap and easy to grow• Popular for micro biology experiments• Host organism for work with recombinant DNA such as POU5F1• Mostly harmless• Rod-shaped
  • 4. Why is POU5F1 valuable?• In 2006, Shinya Yamanaka discovered four transcription factors that was able to generate Induced pluripotent stem cells (Ips Cells) from mouse tissues• In 2007, Shinya and his team were the first group of scientists to discovered only four transcriptional factors that was able to produce iPS cells from human fibroblast cells sox2 POU5F1 Klf4 c-Myc
  • 5. Purpose/Goal• Grow 8 colonies of BL21Star E. Coli at different pH level (4.0-8.0), then run them through TPP and Western Blot to see which E. Coli colony made the most authentic POU5F1 gene at the specific pH level
  • 6. Hypothesis• My hypothesis is the E. Coli grown at pH 7.0 will be able to produce the most authentic POU5F1
  • 7. Materials• BL21 Star E. coli• Centrifuge• Incubator• Sonicator• Nitro Cellulose Paper• Electrophoresis chamber, gel form, and comb• Electrophoresis power supply• Agar gel• Pipettes• Vortex mixer• Centrifuge and cultural tubes (1.5ml, 15ml, 45ml)
  • 8. Materials (buffer, solutions, antibodies)• Phosphate Buffered Saline • Transfer Buffer (PBS) • Primary Antibody• Solid Urea • Secondary antibody• Citrate Phosphate buffer • Horseradish peroxidase• Ammonium sulfate • H20• Kanamycin • BSA Milk• Lysogeny Broth • Standard protein for SDS• methanol PAGE• Chloroform• Tris Glycine SDS Buffer
  • 9. Culturing Bacteria Procedures1. Mix 1 mL of LB (broth) (4.0-8.0) with1mL Kanamycin and pipette it into 15mL cultural tube2. Label each tube with its LB pH level3. Get the BL21star agar plates from the freezer storage4. Use a sterilize tip to touch the surface of each E. coli colony5. Put the tip into the cultural tube and incubate the solution at 37° C for 4 days6. Mix 5mL of kanamycin with 5mL of Lb at each different pH levels7. Discard the tip, then pour the 10mL mixed solution into its corresponding Lb pH tube
  • 10. BL21 Star on Petri Dish
  • 11. Culturing Bacteria Procedures Pt21. Incubate the 8 tubes for 1hr at 37° C2. Transfer 750µL from each cultural tube to 8 new 1.5mL micro centrifuge tubes3. Label the pH levels on the new tubes4. Centrifuge the tubes at 4000g for 20 mins5. Use the vacuum to suck out the SUPT
  • 12. TPP procedures1. Add 1ml PBS and 0.5µL PMSF to each tube2. Sonicate on ice 10 times3. Centrifuge at 9000g, 4° C for 5 min4. Transfer the SUPT to a new 15mL Tube5. Add 100µL DTT6. Add 0.48g of solid urea7. Add .08g (NH4)2SO48. Add 750µL T-Butanol9. Incubate for 1hr at 25° C10. Collect the aqueous phase and transfer it into 5mL tube
  • 13. W&F Protein Precipitation• Prepares for Western Blotting• buffers, detergents, and salt becomes insoluble and change into the organic and aqueous phase• Makes it possible to remove everything except for proteins
  • 14. W&F Protein Precipitation1. Transfer 150µL of each 8. Remove the aqueous sample to a new a 5mL layer tube 9. Add 4 volumes of2. Add 3 volumes of methanol methanol 10. Vortex and centrifuge3. Vortex and then centrifuge11. Remove all supernatant the samples at 15g for 1 12. Add 10µL of 5% SDS to min each sample4. Add 1 volume of 13. Add 10µL Red 2xSB chloroform (sample buffer)5. Vortex and centrifuge 14. Heat each tube in boiling6. Add 3 volumes of water water for 3 minutes7. Vortex and centrifuge
  • 15. Gel Electrophoresis• A method of separating macromolecules based on their size• Samples are loaded into a gel (poly acrylamide) and run through an electric current• DNA is negatively charge so they are pull towards the positive electric currents (bottom) of the gel
  • 16. Gel Electrophoreses Procedure1. Place the gel into the gel box2. Fill the gel box with Tris Glycine SDS Buffer until a thin layer of the buffer covers the gel3. Remove the gel combs and load the standard protein4. Load the samples into the gel5. Place lid on chamber and connect the electrodes to the power supply6. Run the gel on 200V for 30mins
  • 17. 1) Gh2) ,gj3) Hj,
  • 18. Western Blotting• A technique that transfer the proteins from the gel into a nitrocellulose paper so that it can be stained with antibodies to specifically target the protein• The nitrocellulose is then placed with a X-ray film and run through the X Ray
  • 19. Western Blotting Procedures1. Build a “transfer Albumin with non fat sandwich” with the gel powdered milk (liquid and the nitrocellulose form) paper2. Put the sandwich into the electrophoresis chamber and run the electric current3. Place the nitrocellulose on a blocking bag and “block” it with Bovine Serum
  • 20. Western Blot Procedures Cont4. Let the Nitro cellulose incubate at 4 ° C on a shaker5. Rinsed the BSA milk off the nitrocellulose with transfer buffer and add 1.5µL of Primary Antibodies with 300uL transfer buffer6. Let it incubate on a shaker for 3 days at 4 ° C7. Wash off the primary antibodies8. Rinse and remove the primary antibodies9. Add 1.5µL of the second antibodies and incubate for 2hrs 4 ° C10. Rinse the second antibodies11. Add horseradish peroxidase (reporter enzyme) and SuperSignal Chemiluminescent Substrate
  • 21. Results +=
  • 22. Results• Since POU5F1 is 38,571Da we can conclude that it is nearest 37,000 Da and since it was in the lane with the 4.5 pH sample, we can conclude that BL 21 Star E. Coli grown at pH 4.5 is effective at making the most authentic POU5F1 gene
  • 23. Possible Sources of Error• Let the nitrocellulose incubate for too long• Added the wrong amount of a solution that could of skew the results• Didn’t completely wash off the BSA Milk and Antibodies on the nitrocellulose paper
  • 24. Sources• http://www.youtube.com/watch?v=IWZN_G_pC8U• http://www.youtube.com/watch?v=6QYgN-toA1A• http://www.youtube.com/watch?v=v-O103PLhm8• http://www.youtube.com/watch?v=HddVrI2YdZY• http://en.wikipedia.org/wiki/Western_blotting• http://www.piercenet.com/browse.cfm?fldID=8259A7B6-7DA6-41CF-9D55- AA6C14F31193• http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies- application/protocols/western-blotting.html• http://www.piercenet.com/browse.cfm?fldID=01041101• http://www.springerimages.com/Images/RSS/1-10.1385_1-59259-056- X_173-2• http://en.wikipedia.org/wiki/SDS_PAGE• http://en.wikipedia.org/wiki/Gel_electrophoresis
  • 25. Acknowledgments• Dr. Fouad Kandeel• Dr. Kevin Ferreri• Mr. Hyke• Family and Friends