Term
| Native (Non-Denaturing) Gel Electrophoresis |
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Definition
Protein retains native structure and activity
The migration of proteins in native gels is due to both the charge and the size of the protein, and they separate on their charge-to-mass ratio
Buffer usually pH 9, so most proteins are negatively charged
Can determine
Conformation
Quaternary structure (oligomeric state)
Structure/function analysis
Protein-protein interactions
Enzyme assay directly on gel
Analytical purification
Protein modifications
(phosphorylation, glycosylation)
Cannot determine
Molecular weight |
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Term
| Native Gel Electrophoresis (pic) |
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Definition
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Term
| Reversible Uridylylation of PII |
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Definition
Since the PII protein is a trimer of identical subunits and uridylylation occurs at unique sites within each subunit, there are four possible states corresponding to 0, 1, 2, or 3 uridylyl groups per trimer
Lanes 1-4 show a time course of uridylylation of PII
Undetectable by SDS-PAGE |
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Term
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Definition
| Can analyze protein oligomeric state by native gel electrophoresis |
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Term
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Definition
Example – signal transduction protein NRII (nitrogen regulator)
NRII homodimer is two 37.5 kDa wild-type NRII subunits
Hybrid heterodimer is one 77.5 kDa MBP-NRII subunit and one 37.5 kDa NRII subunit
Fusion protein homodimer is two 77.5 kDa MBP fused to NRII subunits
After excision of the different dimer bands, the enzyme was denatured and fractionated by SDS-PAGE to observe the number and size of the subunits |
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Term
| Dimeric Forms of NRII of E. Coli |
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Definition
Wild-type NRII is a homodimer of two 37.5 kDa subunits
MBP-NRII is a homodimer of two 77.5 kDa subunits, consisting of MBP fused to NRII
Hybrid dimer is heterodimer with one NRII and one MBP-NRII subunit, midway between the wild type and the MBP-NRII dimer |
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Term
| Excised Dimer Analysis on SDS-PAGE |
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Definition
| SDS-PAGE analysis of the dimers excised from the gel shown on the previous slide |
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Term
| Pore-Limited Gel Electrophoresis |
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Definition
Nondenaturing (native)
Suitable for the determination of the molecular mass
Resolving gel – gradient from 3 25% polyacrylamide
No stacking gel
Proteins migrate through the gel until they reach a point where the pore size of the medium is too small and they can proceed no further
Thereafter, the individual proteins become focused at the point where the pore size becomes limiting
By including standards on the gel, the molecular mass of the unknown proteins can be estimated |
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Term
| Isoelectric Focusing (IEF) |
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Definition
At a protein’s isoelectric point (pH = pI) it will not move in an electric field
This property is exploited by isoelectric focusing (IEF), which separates proteins by their pI values
A pH gradient is set up within the IEF gel by the inclusion of a variable buffer and ampholytes (low Mw zwitterions)
These, like proteins, have many positive and negative charges and various pI values
During the “pre-running” phase, each of the ampholytes migrates to a position where the pH equals its pI, establishing a pH gradient across the gel. The sample is then introduced, and electrophoresis is continued until the net current is near zero
IEF is useful for determining the pI of proteins
IEF is capable of very high resolution and can be used to detect small changes in proteins, such as covalent modifications and limited proteolysis
However, its use is limited by the high cost of the ampholytes and by the requirement for special equipment
In combination with SDS-PAGE in the two-dimensional electrophoresis method, IEF has played an extremely important role in the identification of numerous proteins |
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Term
| Two-Dimensional Electrophoresis |
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Definition
Combination of IEF and SDS-PAGE
A tube IEF gel is run, and the resulting tube gel is then placed horizontally across the top of the stacking gel of a slab SDS-PAGE gel
The proteins are separated by their pIs in the first (IEF) dimension and according to their molecular mass in the second (SDS-PAGE) dimension
Since both of these techniques are of very high resolution, the cumulative resolution obtained when both are combined is outstanding resolution |
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Term
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Definition
Physiological conditions
Cancer-specific proteins
Heart-disease specific proteins
Heat shock proteins
Mutations/variants
Mutant proteins – excise spot, N-terminal sequencing or mass spectrometry for identification, cloning, etc.
Splice variants
Proteomics
Gene-protein index |
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Term
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Definition
| Native gel -> IEF -> SDS-PAGE |
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