sodium dodecyl sulfate – polyacrylamide gel electrophoresis (sds-page) provides a means to look at...

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

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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