electrophoretic methods: purification and...
TRANSCRIPT
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Protein purification and characterization
Exercise #8 - Electrophoretic methods
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Protein Purification & Characterization
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Electrophoresis
• Characterize (degradation, MW)
• Quantify*
• Determine purity of sample
• Compare proteins from different sources
• Required step for western blotting*
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Gels
Why standard protein electrophoresis
uses Acrylamide and not Agarose?
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Native & denatured PAGE
• Acrylamide/Bisacrylamide
– Inert & thermo-stable
– Control over cross-linking
– Transparent
– Synthetic (reproducible)
• Glycine
– Source for trailing ion (slow mobility)
• Tris-HCl
– Source for leading ion (high mobility)
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?האם אנו נקבל בהכרח הפרדה על פי גודל
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Gel separation efficiency
15% 5%10%
The length of the gel counts!
(Remember Chromatography?)
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Gradient gel is composed of multiple layes of varying % PA
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Native gel
Native gel is w/o SDS – how will it run?
How will protein complex will behave under such condition?
In native gels, protein run along the gel according to their total
charge.
What will happen to a mixture of proteins which are
loaded on such gel? How will they run?
The advantage of native gel is the keeping of the fold.
How can we use this advantage?
Dimerization of
KIR2DL1 in the
presence of Co2
Qing R. Fan et al. JBC 2000
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Troubleshooting “ugly” gels
Smearing
Membrane associated proteinsOverload + uneven casting of gel
Don’t overload!
(use 20ug-40ug/lane max)
Copyright David R. Caprette 2005
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Troubleshooting “ugly” gels
Streaking
Copyright David R. Caprette 2005
Local precipitation
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Troubleshooting “ugly” gels
Gradual fading bands
Copyright David R. Caprette 2005
O.N incubation in DDW
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Troubleshooting “ugly” gels
Skewed run
Copyright David R. Caprette 2005
Spacers not in place – interrupt the electric field
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Troubleshooting “ugly” gels
Bizzare run
Copyright David R. Caprette 2005
Lifting the top hatch (electrode) off while running…
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Isoelectric focusing (IEF)
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2D gels
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Gels are one of the key resources for monitoring the purification process:
• The Degree of protein purification.
•Fractions
• Is the protein present in several fractions?
• Before and after check
• Protein stability over time