Jacobs:Protocol Protein Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Coomassie Staining of Polyacrylamide Gel for Protein

Overview
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method used to separate proteins based on their size. In their native state, proteins fold into unique 3-D structures. In order to separate proteins based on size, they must be unfolded or “denatured” into their primary structure by SDS (detergent). SDS also binds to protein, giving it a negative charge. Negatively charged proteins are separated based on size by running them through a polyacrylamide gel immersed in running buffer and applying an electric field. The negatively charged proteins move toward the positively charged anode, with smaller proteins migrating through the gel faster than larger proteins.

Materials

 * Razor blade
 * Casting plates (spacers)
 * Protein sample
 * Ice bucket with ice
 * 16-well precast tris-glycine polyacrylamide gel (12%)
 * Gel electrophoresis cell
 * 10X tris-glycine running buffer
 * Molecular weight marker
 * SDS sample buffer
 * Power supply
 * Gel separator
 * 1L graduated cylinder
 * Distilled water
 * Pipetman
 * Pipet tips
 * Hamilton syringe and needle for loading gel
 * Polyacrylamide pre-cast gel
 * SimplyBlue™ Coomassie protein stain, 1x pre-mixed solution (Coomassie blue dye binds to proteins (arginine, the aromatic amino acids, and histidine) allowing protein bands to be visualized in polyacrylamide gels.
 * Plastic container
 * Gel separator

Procedure

 * 1) We will use one gel to run protein samples from all groups
 * 2) Put protein sample, molecular weight marker and SDS sample buffer on ice
 * 3) Prepare 1X tris-glycine running buffer
 * 4) Add 100 ml tris-glycine running buffer to the 1L graduated cyliner
 * 5) Add 900 ml distilled water
 * 6) Load pre-cast gel into clamping stand with the comb facing inward
 * 7) Load a spacer into other side of clamping stand
 * 8) Place clamping stand into gel box
 * 9) Add 1X running buffer into inner chamber until gels are completely covered
 * 10) Add 1X running buffer into outer chamber until it is about three-quarters of the way up the gel
 * 11) Pipet 30 ul of your sample into a clean microcentrifuge tube
 * 12) Add 5 ul SDS sample buffer to your sample
 * 13) Boil sample for 3 min using heating block
 * 14) Carefully remove comb from precast gel
 * 15) Load 10 ul of the molecular weight marker into the left-most well in the gel using the Hamilton syringe and needle
 * 16) Load all of your protein sample + SDS sample buffer into an open well
 * 17) Once everyone has loaded their sample into a well, place the lid on the gel box (red wire to red electrode, black wire to black electrode)
 * 18) Run the gel at 100V for ~1hr
 * 19) Ensure that the blue dye front is running down the gel (toward the anode)
 * 20) When gel is finished running, stain gel with SimplyBlue Coomassie protein stain
 * 21) Remove the gel from the gel box and plates using a gel separator
 * 22) Place it in a clean plastic container and cover with ultrapure water
 * 23) Microwave the gel on high for 1 minute until the solution almost boils (do not overheat)
 * 24) Shake the gel on a shaker for 2 minute and discard the water
 * 25) Repeat steps 21 and 22 two more times
 * 26) After the last wash, add 30 ml of SimplyBlue™ (Coomassie protein stain and microwave on high for 45 seconds to 1 minute until the solution almost boils)
 * 27) Shake the gel on a shaker for 10 minutes
 * 28) Wash the gel in 100 ml of ultrapure water for 10 minutes on a shaker
 * 29) Examine gel on a white paper towel for blue-stained protein bands
 * 30) Discard the gel in the Biohazard waste bin

Contact

 * Originally prepared by CRJ-ABC, last updated 8/17/07

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