Term
| Basics of Electrophoresis |
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Definition
Electrophoresis – movement of charged molecules toward an electrode of the opposite charge
When an electric field is placed across a solution containing ions a current develops, charged molecules are separated
Anions move toward the anode (+ electrode)
Cations move toward the cathode (- electrode)
Gel electrophoresis – electrophoresis of charged molecules through a gel meshwork in order to sort them by size or charge |
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Term
| Electrophoresis of Proteins |
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Definition
The separation of proteins gel electrophoresis is influenced by
The charge of the proteins at the buffer pH
The size of the proteins and the frictional resistance as they migrate in the electric field
If these two characteristics compensate for one another, proteins of different charge and size may occasionally migrate at the same rate in an electrophoresis experiment |
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Term
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Definition
SDS-PAGE is used for the routine estimation of protein identification, purity, and quantity
Track the course of purification of proteins for which there is no convenient enzymatic assay (locate the fractions)
Visualize and track protein, contaminants
Resolving power ~1% mass differences |
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Term
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Definition
To simplify the analysis of mixtures of proteins, it is possible to make the separation rely on only the size of the polypeptide chains
Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
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Term
| Sodium Dodecyl Sulfate (SDS) |
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Definition
Each dodecyl sulfate molecule has two negative charges at pH values used for electrophoresis, thus the net charge of the coated polypeptide chains will be much more negative than that of uncoated chains
Identical charge-to-mass ratios
The charge-to-mass ratio will be essentially identical for different proteins because the SDS coating dominates the charge
Thus, the separation of the denatured, detergent-coated polypeptide chains will be due almost exclusively to the size of the polypeptide chains |
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Term
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Definition
Dodecyl sulfate binds strongly to proteins and causes the folded proteins to become denatured due to its negative charge and repulsion into extended “rods” coated with SDS
On average, one dodecyl sulfate molecule will be present for every two amino acids (~1.3g SDS/ g protein) |
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Term
| Detergent and b-Mercaptoethanol |
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Definition
The presence of detergent and the resulting unfolding of polypeptide chains disrupt most of the interactions that hold proteins together (including oligomers), resulting in monomeric polypeptide chains
However, disulfide cross-links between polypeptide chains are not destroyed by heating in the presence of SDS, and thus polypeptide chains so linked will remain covalently bound
These cross-links are eliminated using the reducing agent b-mercaptoethanol which is often added to the sample along with SDS
Some proteins are very resistant to binding SDS and denaturation |
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Term
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Definition
Laemmli buffer
Sample loading buffer – SDS, b-ME, tracking dye, glycerol
Tracking dye
Bromophenol blue helps in loading protein into wells
The high mobility of the tracking dye assures that it will migrate faster than the proteins
When the tracking dye reaches the end of the gel, the electrophoresis is terminated so that the proteins do not run off the end of the gel
Glycerol – weigh down the samples
Aslo may contain a
loading control (actin) |
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Term
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Definition
Electrophoretic separations are performed using a support medium (gel)
Prevents the loss of resolution caused by diffusion and vibrational and convectional disturbances
Retains the separation of different proteins after completion of the experiment
Gel Characteristics
Strong
Hydrophilic to prevent hydrophobic interactions with the proteins
Uncharged
Stable over a range of temperatures, pH, and osmolarity
It should have a carefully controlled and adjustable pore size
Paper and starch gels have played an important role in the development of the technique of protein electrophoresis, although these support medium lack many of the desirable properties
Currently, the supporting medium of choice for almost all protein electrophoresis applications is polymerized acrylamide |
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Term
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Definition
The porosity of the polymerized polyacrylamide gel may be controlled by varying the percentage of acrylamide and/or the degree of cross-linking of the acrylamide chains
Polyacrylamide gels are formed by the polymerization of acrylamide |
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Term
| N,N’-Methylene Bisacrylamide (BIS) |
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Definition
The cross-linking agent of choice for most application is N,N’-methylene bisacrylamide (BIS)
Without BIS, acrylamide forms only linear polymers |
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Term
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Definition
Ammonium persulfate (APS) (-O3S-O-O-SO3-), readily forms unstable SO4- radicals
TEMED (tetramethylethylenediamine) is a tertiary amine that reacts with these radicals to form TEMED free radicals, which in turn react with acrylamide to induce polymerization |
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Term
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Definition
The average size of pores can be controlled by varying
The amount of monomer (acrylamide) used
The degree of cross-linking
Higher degrees of cross-linking results in narrower pores
The degree of cross-linking is generally kept constant, and the percentage of monomer is varied to make gels of different porosity
The pores have a statistical distribution of pore sizes
Small proteins will migrate in the gel with minimal impediment
Larger proteins will be slowed because they cannot pass through all the pores in the gel
Very large proteins and aggregates may not enter the gel
Only proteins that are within a particular size range (molecular weight range, or stokes radius range) will be separated on any given gel |
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Term
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Definition
A gradient gel is a special form of gel in which the degree of porosity is varied continuously from the bottom to the top by creating a gradient in the percentage of the acrylamide
Gradient gels can separate proteins with a wide range of molecular weights |
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Term
| Electrophoresis (Sieving) vs. Gel Filtration Chromatography |
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Definition
Gel filtration: molecules are excluded from the interiors of the gel beads according to their size, so that the largest molecules elute first
Gel electrophoresis: the gel matrix is a mesh-like substances rather than a bead, and therefore, it acts as a sieve, where frictional forces decrease the mobility of larger molecules more than that of smaller molecules
Therefore, large molecules travel more slowly than small molecules |
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Term
| Stacking and Resolving Gels |
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Definition
Acrylamide, BIS, APS, TEMED, Tris, dH2O, SDS
Stacking Gel
Top gel
Stacks proteins into a tight band
5% acrylamide
Resolving Gel
Bottom gel
Separates proteins
10-15% acrylamide |
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Term
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Definition
Proteins be applied to the gel in very small volumes to obtain good separation
A stacking gel is cast directly on top of the larger resolving gel to allow for larger volumes to be loaded
Because the stacking gel is very porous, there is little difference in the mobility of proteins of different size as they migrate through the stacking gel
The proteins are all concentrated into a narrow band at the top of the resolving gel
The stacking gel is polymerized with a lower percentage of acrylamide to ensure high porosity and the resolving gel contains a higher percentage of acrylamide |
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Term
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Definition
Tris – trishydroxymethylaminomethane (2-amino-2-hydroxymethyl-1,3-propanediol) and glycine
The upper and lower reservoir buffer contains Tris pH 8.8 with glycine as the counterion
The stacking gel (in Tris pH 6.8) contains the protein mixture and tracking dye
The pH difference generates an ion gradient via glycine, Cl-, which carry the current |
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Term
| Visualization of Protein Bands |
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Definition
Tightly binding dyes – requires soaking gel in a solution
Coomassie brilliant blue R-250
A dye closely related to that used in the Bradford assay for total protein concentration
Binds strongly to proteins (aromatics, arginine, histidine)
Destain with methanol
Not very sensitive
Silver staining
Silver atoms bind very tightly to proteins and produce black or purple bands (as in photography)
50-100X more sensitive
More expensive, longer to perform
Toxic
Fluorescence
2,2,2-Trichloroethanol (TCE) – reacts with tryptophan under UV
Can be used downstream (i.e. western blotting)
The most common groundwater contaminant in the U.S.
Radioisotope detection (autoradiography)
X-ray film
35S in methionine, 32P, 33P (X-ray)
14C, 3H (fluorophores)
Antibody detection (Western Blotting) |
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Term
| Coomassie-Stained Slab SDS-PAGE Gel |
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Definition
Protein from SEC column
An aliquot from every third fraction was run, from left to right on the gel
A prominent band is observed samples 4-9 |
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Term
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Definition
Uneven electric field
Overheating
Incorrect polymerization |
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Term
| Quantification of Protein Bands |
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Definition
Markers
Standard curve
Stained gels can be scanned in a densitometer to quantify the relative amounts of the various proteins bands (concentration)
Individual proteins bind to the dye distinctly, and in the absence of specific knowledge on the binding of each protein to the dye, quantification of stained bands is only approximate (like DC protein assay, Bradford assay, gel filtration Mw determination) |
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Term
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Definition
Relative mobilities of proteins, Rf, is determined as the ratio of the distance each protein migrates to that of the tracking dye
The high charge-to-mass ratio of the dye causes it to migrate near to the electrophoresis front and ahead of the proteins
Different gels can be compared due to the top of gel/tracking dye ratio |
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Term
| Molecular Weight Estimation (Standard Curve) |
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Definition
Proteins of known molecular mass are run on the gel simultaneously with the unknown polypeptide, and the relative mobility (Rf) of each species is determined
The relative mobility is calculated by dividing the distance the protein migrates by the distance the tracking dye migrates |
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Term
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Definition
Photograph
Store on computer
Publications
Gel drying
Lab notebook
Cover gel with plastic wrap and dry the gel in a gel dryer
Dry in piece of cellophane while it slowly dries at ambient conditions |
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