Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide Gel Electrophoresis (PAGE) (Overview)

The goal of polyacrylamide gel electrophoresis, or PAGE, is to separate proteins within a protein lysate according to size, shape, and/or charge.

To accomplish this, protein lysates are loaded onto a gel matrix. The matrix is made up of holes through which proteins can pass.

The smaller the protein, the faster it will pass through the holes in the gel matrix.

The bigger the protein, the longer it will take to go through the gel matrix.

Gel Preparation orientation of electrodes

In PAGE, to gel solution containing the acrylamide compounds, catalyst, and polymerizing agent is prepared and quickly poured between two plates anchored vertically in a cassette.

A small plastic comb is placed at the top to create wells where the protein lysates can be loaded.

As the liquid solution hardens, a flexible thin gel forms between the glass plates.

Once the gel completely polymerizes, the gel cassette is inserted into a chamber, which contains buffer to allow the conduction of current through the gel, with the negative electrode positioned at the top of the gel chamber, and the positive electrode electrode positioned at the bottom of the chamber.

negative at top positive at bottom

Denaturing Gel and SDS

In a denaturing gel, proteins are assessed in a denatured, or unfolded, state and separated in the gel matrix according to their mass only.

To accomplish this, sodium dodecyl sulfate, or SDS, an anionic detergent, is added to denature proteins and uniformly coat them with a negative charge.

This means that proteins with similar mass but different shapes migrate at similar rates in an SDS page.

By the same token, proteins with similar mass but different overall charges also migrate through the gel matrix at comparable rates.

During SDS PAGE, SDS is added to the gel solution, protein samples, and running buffer to ensure that proteins remain negatively charged and unfolded throughout the procedure.

Sample Buffer

Before running a polyacrylamide gel, researchers need a way to easily load the protein samples into the wells, which are immersed in running buffer, and monitor the proteins as they migrate through the gel since proteins are mostly colorless.

To do this, a dye solution called sample buffer is added to the protein lysates. Sample buffer contains an additive such as glycerol, which makes the sample buffer denser than the surrounding running buffer, causing the samples to sink to the bottom of the wells rather than mixing with the surrounding buffer. Sample buffer also contains a negatively charged dye, typically bromophenol blue, used to monitor the progress of electrophoresis. Tracking or loading dyes, as they are often referred to, have low molecular weights and migrate ahead of most proteins within the protein sample.

Protein Denaturation and Disulfide Bond Disruption what does it

Once the protein lysate and sample buffer mixture are ready, the protein samples are heated in sample buffer for a short time to facilitate protein unfolding for SDS PAGE.

In addition, sample buffer contains beta-mercaptoethanol and DDT to disrupt disulfide bonds, further assisting in protein unfolding by disrupting quaternary (and tertiary) structure.

Protein Ladder

asses molecular weight, containing several pre-stained proteins of known size, is loaded and run along with the experimental samples.

The use protein ladders with pre-stained proteins increases visibility and facilitates monitoring of protein migration through the gel.

After loading the protein lysates and ladder, the gel is run by connecting the chamber to a power supply