When preparing an SDS-PAGE, a) Two gels are prepared independently and one gel i

Question

When preparing an SDS-PAGE,

a) Two gels are prepared independently and one gel is casted on top of the other. Name the gel that is casted on the bottom. (2pts)

b) Name the gel that is casted on top (where the samples are loaded). (2pts)

c) What is plotted on the axis for a standard curve derived from an SDS-PAGE protein ladder? (2pts)

i. Y-axis:

ii. X-axis:

d) Which gel must be overlaid with water? (2pts) Why? (2pts)

e) What dye was used to stain the proteins after the electrophoresis was completed? (2pts)

f) Why are the protein samples heated prior to loading them into an SDS-PAGE? (2pts)

Explanation / Answer

When preparing an SDS-PAGE,

a) Two gels are prepared independently and one gel is casted on top of the other. Name the gel that is casted on the bottom. (2pts)

It is called the Running Gel

b) Name the gel that is casted on top (where the samples are loaded). (2pts)

It is called the Stacking Gel

c) What is plotted on the axis for a standard curve derived from an SDS-PAGE protein ladder? (2pts)

i. Y-axis: Molecular Weight (Da)

ii. X-axis: Distance travelled down gel (mm)

d) Which gel must be overlaid with water? (2pts) Why? (2pts)

The top gel must be overlaid with water

Immediately after pouring the gel mix, it must be overlaid with water-saturated butanol to an additional height of 0.5 cm or so (butanol is the top layer in the stock container). Adding butanol to a single cassette will drive the acrylamide mix down, raising the level in the others, so care must be taken to distribute the butanol equally among the cassettes. The purpose of butanol is to produce a smooth, completely level surface on top of the separating gel, so that bands are straight and uniform. Butanol holds very little water in solution, forming a neat layer on top, which is why we use it. Water would make an effective overlay but would mix with the acrylamide solution, diluting it. In fact, the butanol we use is saturated with water so that it does not dry out the gel mix.

e) What dye was used to stain the proteins after the electrophoresis was completed? (2pts)

Following electrophoresis, the gel may be dyed (for proteins, most commonly with Coomassie Brilliant Blue R-250; for nucleic acids,ethidium bromide; or for either,silver stain), allowing visualization of the separated proteins

f) Why are the protein samples heated prior to loading them into an SDS-PAGE? (2pts)

Proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or2-mercaptoethanol (beta-mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE.

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