Lab 4 - SDS/PAGE (Gel Electrophoresis)

Why was SDS added to our protein samples prior to electrophoresis?

(1) Coat proteins with negative charge (proteins will run toward anode) (2) Denature proteins (unfold and make proteins more linear so run true according to molecular weight)

What is the most common matrix for running DNA?

Agarose

What types of gels can be used for gel electrophoresis?

Agarose or polyacryalmide

After we run our fractions (WC, Cytoplasmic, and NC) and stain them, we look for what?

Blue band of our unknown near the approximate molecular weight of FAM171B (90 kD)

What stain did we use in lab; what does it stain?

Coomassie; stains/binds ALL proteins and stains them blue.

Why is agarose an OK matrix to use for running DNA?

DNA has an inherent negative charge so will move to the positively charged anode (on bottom)

During loading, if any sample spilled over to a neighboring lane, how could that affect your interpretation of the results?

Each lane is supposed to contain the same amount of protein so if one lane spilled into another, that lane would have more proteins than the other and skew our observations.

Gel electrophoresis uses ________ to draw charged macromolecules through some kind of gel matrix.

Electricity

Why use the ladder in this experiment?

In order to compare the unknown fragments with the known ones and make a reasonable estimate of the unknown fragment size

What control did we use in our SDS/PAGE experiment?

Known standards (known molecular weights) alongside our unknown fragments. (also known as the ladder)

The cathode is ________ charged and located where?

Negatively; on top

Would proteins run well on an agarose gel?

No because proteins don't have a negative charge so won't move through the gel (no separation would occur)

Is FAM171B a nuclear or cytoplasmic protein?

Nuclear protein

What is the most common matrix for running proteins?

Polyacrylamide

The anode is ________ charged an located where?

Positively; on bottom

What is the purpose of gel electrophoresis?

Separate subcellular macromolecules based generally upon their size or molecular weight.

Upon completion of electrophoresis, how should the gel be separated?

Smaller molecules positioned near the bottom of the gel while larger ones will remain nearer to the top.

What moves through the gel faster to the bottom: smaller molecules or larger molecules. Explain?

Smaller molecules; Pores in wells get smaller with distance so smaller molecules can sneak through the gel pores more easily.

Proteins nor DNA has an inherent color, so after electrophoresis, what must we do?

Stain them macromolecules in order to visualize them in the gel.

Gel electrophoresis allows for separation of molecules based on what?

Their molecular weight

Why was it important to add SDS to our samples and gel?

To (1) coat proteins with negative charge (2) denature proteins so they run "true"

Proteins require an extra step as they exist within the cell with a ________ charge. What is this extra step and why is it done?

Variable; additon of SDS prior to electrophoresis; (1) Coat proteins with negative charge (2) Denature proteins so unfolded and near more linear structure to run more "true" according to their molecular weight.

How did we ensure equal loading of protein in each gel lane?

We performed a protein assay in lab 3 and calculated, using the standard curve, the concentration of each fraction to add.

DNA molecules only move from the negative ________, on (top/bottom) to the positive ________, on (top/bottom).

cathode; top; anode; bottom