Identifying POU5F1Duy NguyenBiomedMr. Hyke

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

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

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 cellssox2POU5F1Klf4c-Myc

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

Hypothesis• My hypothesis is the E. Coli grown at pH 7.0 will

be able to produce the most authentic POU5F1

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)

Materials (buffer, solutions, antibodies)

• Phosphate Buffered Saline (PBS)

• Solid Urea• Citrate Phosphate buffer• Ammonium sulfate• Kanamycin• Lysogeny Broth• methanol• Chloroform• Tris Glycine SDS Buffer

• Transfer Buffer• Primary Antibody• Secondary antibody• Horseradish peroxidase• H20• BSA Milk• Standard protein for

SDS PAGE

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 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

BL21 Star on Petri Dish

Culturing Bacteria Procedures Pt2

1. 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

TPP procedures

1. 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 DTT 6. Add 0.48g of solid urea7. Add .08g (NH4)2SO4

8. Add 750µL T-Butanol9. Incubate for 1hr at 25° C10.Collect the aqueous phase and transfer it into

5mL tube

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

W&F Protein Precipitation

1. Transfer 150µL of each sample to a new a 5mL tube

2. Add 3 volumes of methanol

3. Vortex and then centrifuge the samples at 15g for 1 min

4. Add 1 volume of chloroform

5. Vortex and centrifuge6. Add 3 volumes of

water7. Vortex and centrifuge8. Remove the aqueous

layer9. Add 4 volumes of

methanol10.Vortex and centrifuge11.Remove all

supernatant12.Add 10µL of 5% SDS

to each sample13.Add 10µL Red 2xSB

(sample buffer) 14.Heat each tube in

boiling water for 3 minutes

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

Gel Electrophoreses Procedure

1. 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 gel

5. Place lid on chamber and connect the electrodes to the power supply

6. Run the gel on 200V for 30mins

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2) ,gj

3) Hj,

4)

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

Western Blotting Procedures

1. Build a “transfer sandwich” with the gel and the nitrocellulose 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 Albumin with non fat powdered milk (liquid form)

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 ° 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

Results

+

=

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

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

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

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