SDS-PAGE Image Analysis

What is the approximate mass of a Dalton or hydrogen atom

1.66 × 10^-24 grams

What is the average mass of an amino acid?

110 daltons

An ___ that ___ and ___ proteins

anionic detergent, solubilizes, denatures

2-mercaptoethanol is a ___ agent that ___ to achieve ___

reducing, cleaves disulfide bonds, complete protein unfolding

Boiling ensures the ___ of the proteins and that they are completely ___

total denaturation, completely linear

Compare polyacrylamide gel to agarose gel

Polyacrylamide gel has a tighter matrix or smaller pores, making it more efficient than agarose gel

Average size of amino acid? nucleotide pair? 1 kilobase of DNA

• Average amino acid = 110 daltons

• Average nucleotide pair = 649 daltons

• 1 kilobase of DNA = 650 kD

Proteins start separating at the ___ gel

Running/resolving

Stacking gel is just for ___, so ___ pores

concentrating the sample, wider

Describe the pore size of PAGE, 3% agarose, and 1% agarose

1. Polyacrylamide gel: 20–200 nm pores

2. 3% agarose: 40–80 nm pores

3. 1% agarose: 200–1200 nm pores

What happens if we use DNA for SDS-PAGE?

There is poor gel quality and less defined resolution of bands

How do we validate the identity of proteins with similar or same molecular weights?

Protein sequencing

What is also still needed to confirm protein identity using PAGE

Western blot analysis

SDS-PAGE could be applied to DNA when?

1. DNA are shorter sequences or fragments

2. microsatellites

3. microRNA

Describes the migration of a sample through a gel from its initial to final position?

Migration distance

To determine the molecular size and weight of protein bands in a given sample, you have to prepare a ___ based on the molecular weight of the ___

standard curve, protein standard or protein ladder

What does migration distance describe

Mobility or how long or fast they travel from cathode to anode

The x- and y- axis for SDS PAGE

x-axis: migration distance, y-axis: log molecular weight

Formula for Rf or retention factor or relative migration distance

migration distance of the sample or band/migration distance of the dye front

What is the dye front?

Bromophenol blue

Where do we start in measuring migration distance?

Start from running gel because this is where the actual separation happens

Contrast when to use the actual migration distance versus relative migration distance

1. Actual → a singular gel

2. Relative → multiple gels, can help you determine variations especially when you have replicates

Where is the dye front positioned?

Measured/positioned at the

bottom part of the gel beyond the smallest protein standard

The x-axis for SDS-PAGE Image Analysis could be either?

• migration distance

• relative migration distance if analyzing multiple gels

The y-axis for the SDS-PAGE Image Analysis

log(kDa) or log(MW)

Why must we use log(MW)?

If you use the actual MW, you won’t be able to generate a standard curve with an acceptable R² value

Provide the variables for the line equation y = mx + b

MW(log) = m(d*) + b