protein localization in mammalian cells
TRANSCRIPT
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Cynthia Becerra, Corrina Di Pirro, Christina Fennell, Leslie Galvan, Emma Inhorn, Ashley Klesken, Queena
Luu, Michelle Tankimovich, Evelyn Torres
PROTEIN LOCALIZATION IN MAMMALIAN CELLS
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OBJECTIVE • Compare the localization of proteins in mammalian cells • Cells used: • Human embryonic kidney cells and mouse embryonic
fibroblast cells • Proteins used: • ZFP568 and GALT
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OBJECTIVE CONT. • Proteins are chains of amino acids determined by DNA • Significance the specific locations of proteins are
essential to an organism’s proper function • ZFP568 basic development • GALT sugar metabolism that affects the development
of organs
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PREVIOUS KNOWLEDGE • We knew what type of cells we worked with • We knew where the proteins were supposed to be located • ZFP568 nucleus • GALT Golgi body
• We did not know which proteins we worked with specifically
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UNKNOWN KNOWLEDGE • Protein A and Protein B
• PLAN TO COMPLETE GOAL • Once placing the proteins into the cells • Upon analyzing the cells based on the location of
the proteins • the specific proteins can be identified
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MODEL CELL
http://www.animalport.com/img/Animal-Cell.jpg
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• Circular DNA that contain genes for survival
ZFP 568 or GALT start reading DNA here
KAN stop reading DNA here
With Proteins
XhoI
HindIII
Start reading DNA here
Stop reading DNA here
XhoI HindIII
Control Group GFP
KAN
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Start Centrifuge Lysed
Separate Purify
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• Transfection: Putting DNA into mammalian cells
• Mix DNA in media with Lipofectamine
• Allows DNA transportation in a liposome • A vesicle made of a phospholipid bilayer
Transfection in Mouse and Human Kidney Cells
• Cells take up DNA and express the proteins • GFP-alone • GFP-ZFP568 • GFP-GALT
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WHY NIH3T3 AND HEK293T? • NIH3T3 Cells (Mouse Embryonic Fibroblasts) • Have more cytoplasm
• HEK293T Cells (Human Embryonic Kidney) • Good for transfection
• Commonly used by scientists; easy to transfect • Mislocalization of proteins commonly signify disease • Ex: Cystic Fibrosis (CF)
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We stained each sample with three different stains: 1. GFP (Green Fluorescent Protein)
2. DAPI (Blue, stains nuclei)
3. Phalloidin (Red, stains cytoskeleton)
THE MAGIC OF STAINING
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• We used special pipetting techniques to stain cell structures
• Kept cells covered from light and exposure to air
• Secured with cover slip
HOW DID WE DO IT?
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HEK293T Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
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HEK293T Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
GFP- GALT
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HEK293T Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
GFP- GALT
GFP- Alone
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HEK293T Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
GFP- GALT
GFP- Alone
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NIH3T3 Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
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NIH3T3 Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
GFP- GALT
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NIH3T3 Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
GFP- GALT
GFP- Alone
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NIH3T3 Cells
Bright Field GFP Phalloidin DAPI Merged
GFP- ZFP568
GFP- GALT
GFP- Alone
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- The Zinc Finger Protein 568 affects the transcription of DNA in the nucleus
- Galt affects the sugar metabolism and the Golgi in the cell
So why is it important? - Mislocalization of protein leads to diseases and its important for scientific understanding
SIGNIFICANCE
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Special Thanks To:
-CURIE Academy Faculty -Professor Shen and Professor Land -Sara Hernandez -Angela Pring-Mill, Most Helpful TA Ever