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Gel Electrophoresis
Gel Electrophoresis
Chamodhi Ranathunga
Dictyostelium discoideum Metabolism of
Inositol Pyrophosphate by Gel Electrophoresis
Purpose:
- Analyze the metabolism of insositol pyrophosphate in Dictyostelium discoideum
- Development revealed less dramatic increase of IP8 through PAGE than Strong anion exchangechromatography (Sax-HPLC)
What is Gel Electrophoresis?
Results
Methods/Techniques
PAGE anaylisis of IP during D. Discideum development
PAGE (polyacrylamide gel electrophoresis)
- compare wild type (WT), ip6k null (IP6 Kinase), and phospholipase
- Toluidine vs DAPI (photo bleaching)
Enzymology
- confirm nature of IP as genuine
Incubated acidic extract at 90C for 10 minutes before neutralization
- degradation of IP7 and IP8 result in formin IP6
Spectrometry- m/z range 500-1000
- IP7 peaks: 738.822 & 760.793 m/z
- IP8 peaks: 840,590 & 818.627 m/z
Method
PAGE IP6, IP7 & IP8
reveal 3 major bands
Spectromery
Enzymology WT incubated with:-
A: phytase treatment
B:recombinant DDP1
C: acid at high temperature
1. Each mixture of different size DNA sample in separate wells on gel at negative end of gel.
-DNA dyed with fluorescent dye
2. When current is turned on, DNA molecules move toward positive side.
- Phosphate groups have negative charge
3. Put in ultraviolet light so that DNA molecules visible as pink bands
Procedure used to separate DNA fragments (nucleic acids/proteins)
- Electrical current that passes through gel
- move from negative to positive
- phosphate group negative charge
- separate according to size
- short segments move further than long ones
Work Cited
Dictyostelium discoideum
Dictyostelium discoideum: type of amoeba that metabolizes inositol pyrophosphate
- IP: regulate diverse biological process
- insulin signalling
- inositol: atom rings in nucleus; alcohol w/ 6 possible isonomers
- pyrophosphate: 2 pho
- http://www.dnalc.org/resources/animations/gelelectrophoresis.html
- Pisani, Francesca, Thomas Livermore, Giuseppina Rose, Jonathan Robert Chubb, Marco Gaspari, Adolfo Saiardi, and Thierry Soldati. "Analysis of Dictyostelium Discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis." PLoS ONE (2014): E85533. Web. 8 Jan. 2015. <http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085533>
Conclusion
- Used the technology to resolve and visualize inositol phosphates (which was to characterize in vitro enzymatic reaction)
- Sax-HPLC method is inferior to that of PAGE
- inexpensive
- resolve different isomeric species
- genetic system surpassing S. cerevisae model because of high complexity and greater similarity