POU5F1 is a transcription factor required for embryo development. The document describes growing E. coli colonies at different pH levels to determine which pH produces the most authentic POU5F1 protein when used to express the POU5F1 gene. Western blotting was used to analyze protein expression, and showed that E. coli grown at pH 4.5 expressed the most POU5F1, near the expected molecular weight of 38,571 Da. Possible sources of error include incubating the nitrocellulose membrane for too long or incomplete washing of antibodies.

1. Identifying
POU5F1
Duy Nguyen
Biomed
Mr. 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 Procedures
1. Mix 1 mL of LB (broth) (4.0-8.0) with1mL Kanamycin
and pipette it into 15mL cultural tube
2. Label each tube with its LB pH level
3. Get the BL21star agar plates from the freezer storage
4. Use a sterilize tip to touch the surface of each E. coli
colony
5. Put the tip into the cultural tube and incubate the
solution at 37° C for 4 days
6. Mix 5mL of kanamycin with 5mL of Lb at each different
pH levels
7. 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 Pt2
1. Incubate the 8 tubes for 1hr at 37° C
2. Transfer 750µL from each cultural tube to 8 new 1.5mL
micro centrifuge tubes
3. Label the pH levels on the new tubes
4. Centrifuge the tubes at 4000g for 20 mins
5. Use the vacuum to suck out the SUPT

12. TPP procedures
1. Add 1ml PBS and 0.5µL PMSF to each tube
2. Sonicate on ice 10 times
3. Centrifuge at 9000g, 4° C for 5 min
4. Transfer the SUPT to a new 15mL Tube
5. Add 100µL DTT
6. Add 0.48g of solid urea
7. Add .08g (NH4)2SO4
8. Add 750µL T-Butanol
9. Incubate for 1hr at 25° C
10. 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 Precipitation
1. Transfer 150µL of each 8. Remove the aqueous
sample to a new a 5mL layer
tube 9. Add 4 volumes of
2. Add 3 volumes of methanol
methanol 10. Vortex and centrifuge
3. Vortex and then centrifuge11. Remove all supernatant
the samples at 15g for 1 12. Add 10µL of 5% SDS to
min each sample
4. Add 1 volume of 13. Add 10µL Red 2xSB
chloroform (sample buffer)
5. Vortex and centrifuge 14. Heat each tube in boiling
6. Add 3 volumes of water water for 3 minutes
7. 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 Procedure
1. Place the gel into the gel box
2. Fill the gel box with Tris Glycine SDS Buffer until a thin
layer of the buffer covers the gel
3. Remove the gel combs and load the standard protein
4. Load the samples into the gel
5. Place lid on chamber and connect the electrodes to the
power supply
6. Run the gel on 200V for 30mins

17. 1) Gh
2) ,gj
3) Hj,

19. 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

20. Western Blotting Procedures
1. Build a “transfer Albumin with non fat
sandwich” with the gel powdered milk (liquid
and the nitrocellulose form)
paper
2. Put the sandwich into the
electrophoresis chamber
and run the electric
current
3. Place the nitrocellulose on
a blocking bag and “block”
it with Bovine Serum

22. Western Blot Procedures Cont
4. Let the Nitro cellulose incubate at 4 ° C on a shaker
5. Rinsed the BSA milk off the nitrocellulose with transfer
buffer and add 1.5µL of Primary Antibodies with 300uL
transfer buffer
6. Let it incubate on a shaker for 3 days at 4 ° C
7. Wash off the primary antibodies
8. Rinse and remove the primary antibodies
9. Add 1.5µL of the second antibodies and incubate for
2hrs 4 ° C
10. Rinse the second antibodies
11. Add horseradish peroxidase (reporter enzyme) and
SuperSignal Chemiluminescent Substrate

23. Results
+
=

24. 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

25. 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

26. 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

27. Acknowledgments
• Dr. Fouad Kandeel
• Dr. Kevin Ferreri
• Mr. Hyke
• Family and Friends