Lab 3: SDS-PAGE

Sample Buffer

• what it is:

  • mixed with our sample at the end of Lab 2

• its components:

  • - charged SDS detergent

  • DTT

  • glycerol

  • tracking dye

SDS

• Disrupts protein folding, which causes the proteins to denature and become rod-shaped – meaning that the movement of the protein through the gel does not depend on the protein’s shape.

• SDS also coats the polypeptides with negative charges to increase the speed at which the proteins will migrate towards the positively charged electrode.

  • Note that most proteins do already carry a negative charge in buffers with a basic pH, but SDS will also mask any existing charges so that all the proteins become negatively charged

• 2 versions

  • SDS-PAGE: denatures the proteins structure

  • SDS-Native: omits denaturing chemicals and reducing agents in the Sample Buffer and the gel, thus conserving interactions between polypeptide subunits

DTT

• beta mercaptoethanol

• reducing agent which will break the strong disulfide bonds within the protein.

  • SDS does not break disulfide bonds

Tracking Dye

• in the sample buffer

• allows us to follow the progress of electrophoresis.

• charged blue dye migrates through the gel just ahead of the smallest proteins.

  • therefore, the process of electrophoresis can be tracked by visually following the blue dye

Coomassie Blue

• If you were interested in immediately seeing alllllllllll the proteins present in your cell lysate, you could stain the gel with Coomassie Blue.

• However, this has limited usefulness, as it is almost impossible to know which band corresponds to any particular protein of interest.

• Note: many proteins can have the same molecular weight, so each band could actually be hundreds of different proteins that ended up at the same spot in the gel.

Molecular Weight Marker

• if proteins of known molecular weights are ran alongside the protein samples in the same gel, their migration distances can be plotted as a function of log molecular weight to produce a standard curve (straight line).

• The distances migrated by sample proteins can be compared to the graph to accurately estimate the molecular weights

SDS-PAGE vs. Native-PAGE

• SDS-PAGE:

  • denatures the proteins structure

• Native-PAGE

  • omits denaturing chemicals and reducing agents in the Sample Buffer and the gel, thus conserving interactions between polypeptide subunits

Protein Electrophoresis Theory

• Electrophoresis is a common method for separating charged molecules in an electric field.

• Molecules are driven through a cross-linked matrix (gel typically made of polyacrylamide) which acts as a molecular sieve, allowing differential migration of molecules based on size/molecular weight.

• Small molecules fit easily though the pores in the gel and migrate rapidly, whereas larger molecules become entangled in the gel matrix and therefore move more slowly

Steps (part 1)

• Place 2 tubes of cell lysate into a hot water bath for 5 minutes to denature the proteins, then centrifuge to eliminate bubbles

• Set up/load the Acrylamide Gel in the Electrophoresis Tank:

  1. Wearing gloves, place a black clamp into the left chamber of the tank

  2. Pour running buffer into the chamber with the clamp, so it is level with the silver electrode

  3. Place the acrylamide gel into the chamber in front of the clamp, and position so that the lower slanted plastic plate faces you and the front of the tank

  4. Pull the level on the clamp forward to lock the gel into place.

  5. Using a P20, load your gels into each lane

  6. Fit the lid into the unit, and attach to power supply. Bubbles along the top of the gel indicates that it is running.

• Allow set up to run until the tracking dye is close to the bottom of the gel. Then stop the unit, disconnect the electrodes, and remove the lid.

• Pour the buffer out of the unit, unlock the clamp, and remove the gel.

Protein Transfer from Gel → Nitrocellulose

• In preparation for Lab 4, when you will be performing a western blot, the proteins in the gel must be transferred to a piece of nitrocellulose membrane.

• This is achieved by stacking the gel on top of the nitrocellulose membrane and using a Bio-Rad Trans-Blot Turbo Machine to apply a current.

• This moves the negatively charged proteins out of the gel and onto the nitrocellulose membrane.

Step 1

• Place 2 tubes of cell lysate into a hot water bath for 5 minutes to denature the proteins, then centrifuge to eliminate bubbles

Step 2

• Set up/load the Acrylamide Gel in the Electrophoresis Tank:

  1. Wearing gloves, place a black clamp into the left chamber of the tank

  2. Pour running buffer into the chamber with the clamp, so it is level with the silver electrode

  3. Place the acrylamide gel into the chamber in front of the clamp, and position so that the lower slanted plastic plate faces you and the front of the tank

  4. Pull the level on the clamp forward to lock the gel into place.

  5. Using a P20, load your gels into each lane

  6. Fit the lid into the unit, and attach to power supply. Bubbles along the top of the gel indicates that it is running.

Step 3

• Allow set up to run until the tracking dye is close to the bottom of the gel. Then stop the unit, disconnect the electrodes, and remove the lid.

Step 4

• Pour the buffer out of the unit, unlock the clamp, and remove the gel.

Step 5

• Use the gel knife to separate the plastic plates and expose the gel. Once out, our running buffer onto the gel so that it doesn’t dry out. Trim accordingly

Step 6

• Layer, in order, filter paper, nitrocellulose, gel, and a second layer of filter paper. Roll with small roller to ensure complete removal of all bubbles

Step 7

• Place gel in tray, slide into drawer and into Turbo-blot machine and let run

Step 8

• After the layers have ran, place the nitrocellulose in a dish with TBS-tween.