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Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE)
Provides a means to look at all the proteins in a cell simultaneously
Electrophoretic mobility of a protein depends on its molecular weight
What principles govern the separation of proteins by SDS-PAGE?
How does one prepare and run SDS-PAGE gels?
How are SDS-PAGE gels analyzed?
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Heat
Proteins are prepared by boiling in the presence of SDS
SDS binding imparts a constant charge/mass ratio to the protein
-
SDSSodium dodecyl sulfate
Free radicals initiate a vinyl polymerization reaction
Caution: Unreacted acrylamide and bisacrylamide monomers are neurotoxins – polyacrylamide is inert
Gels are crosslinked matrices of acrylamide polymers
"Discontinuous" gels have stacking and running gels with different compositions
Protein migration varies greatly in the stacking and running gels
Stacking gel0. 125 M Tris-HCl, pH 6.8 5% acrylamide* Larger pores, lower ionic strength
Running (resolving) gel
0. 375 M Tris-HCl, pH 8.8 12% acrylamide* Smaller pores, higher ionic strength
*Investigators adjust the acrylamide concentration to manipulate the gel pore sizes
pKa = 2.31pKa = 9.60
Ionization of glycine in running buffer is key to protein separation
Both amino and carboxyl groups are charged in stacking gel (pH 6.8)
Amino group loses some positive charge in the running gel (pH 8.8)
Zwitterionic form
Glycine is a major component of the gel running buffer
Running buffer: Tris-glycine-SDS, pH 8.3
Protein samples are loaded into wells
Bromophenol blue in sample buffer will act as a tracking dye
At the start:
pH 6.8
pH 8.8
____
+
Chloride ions in the stacking gel buffer move rapidly to positive pole
Proteins are negatively charged and run at various rates through the stacking gel
Glycine moves more slowly than the proteins, since very few glycine molecules have a negative charge
Voltage is applied
Glycine molecules enter stacking gel
Proteins "stack up" at the interface between the two gels
pH 8.8
+
Differential migration of chloride and glycine ions sets up a potential difference that helps to concentrate proteins
+
Glycine (now negatively charged) moves more rapidly than proteins at the pH of the running gel
Proteins are resolved by size in the running gel
Migration of proteins (invisible) in running gel is inversely proportional to their log(MW)
Glycine amino groups lose a proton as they enter the running gel
What principles govern the separation of proteins by SDS-PAGE?
How does one prepare and run SDS-PAGE gels?
How are SDS-PAGE gels analyzed?
Freshly prepared solution of 10% APS is used
TEMED is used undiluted AND added last
Chemicals required for SDS-PAGE gels
Larger plate has precisely milled spacers at either edge that will generate a 1 mm separation between the assembled plates
Gel will be formed between two glass plates
Second plate is shorter, but has the same width as the spacer plate
Plate is VERY fragile!
Make sure plates are CLEAN before proceeding!
Setting up the casting frame
Gates on frame are open: No pressure on plates
Large plate is in back (should be able to read letters)
Feet of casting stand AND bottom edges of plates are flush with the bench surface (important for seal)
Closing the gates on the frame seals the plates together
Clip the assembled casting frame into the casting stand (can accommodate two gels)
Spongy pad at the bottom of the stand contributes to a tight seal
Test for potential leaks with deionized water
Pour the water out after you have finished the test
Mix the chemicals in a test tube
Add the TEMED last and work quickly once it’s added!
Pipet acrylamide mixture to the top of the frame gates
Avoid air bubbles as much as possible (O2 inhibits polymerization)
Overlay the acrylamide with a thin layer of deionized water
The interface between the running gel and water will disappear as the gel polymerizes, but it will reappear as a sharp boundary when polymerization is
complete
A sharp interface appears when polymerization is complete
Remove the unpolymerized upper layer
Combine the components of the stacking gel, adding catalysts last
Pipette the solution on top of the running gel, leaving some room for the sample comb – BE QUICK!
Insert the sample comb into the stacking gel solution
Be careful to avoid trapping bubbles! (may need to reposition the comb)
Time to run the gel!
Remove the gel from the casting frame
Next: position the gel in the electrode apparatus
Upper edge of the short plate should be flush with the gasket
Remove the sample comb after the gel has polymerized
Add running buffer to the upper and lower reservoirs
Load the samples
Place a loaded gel loading tip at the bottom of a sample wellSlowly expel the sample, avoiding air bubbles
Connect the gel to the power supply
Monitor the progress of the tracking dye
Turn off the power before the tracking dye runs off the gel!
What principles govern the separation of proteins by SDS-PAGE?
How does one prepare and run SDS-PAGE gels?
How are SDS-PAGE gels analyzed?
Gels must be stained to visualize proteins
Exceptions are prestained protein standards that have covalently attached chromophores (right
lane)
Proteins are visualized with Coomassie Brilliant Blue G-250
Gels are rinsed with water several times to remove gel chemicals and are then incubated with Simply Blue® a colloidal suspension of Coomassie Blue G-250
BSA – 78K
CA – 45.7K
SBTI - 32.5K
B-gal 132K
Log 10
mol
ecul
ar w
eigh
t
Distance migrated (mm)
Sizes of proteins can be calculated by comparing their migration to those of marker proteins