A glossary of electrophoresis terms

Electrophoresis is motion in an electric field, through a matrix that makes the motion informative. The words on this page are the ones that decide whether a notebook sentence is physically true. The photograph is an agarose gel on a UV transilluminator under an orange shield, which is one apparatus these terms describe. How that particular gel is planned and read is the pillar note, agarose gel electrophoresis for DNA. Protein lanes use the same field words and a different matrix, as in reading a protein gel.

Anode and cathode

The anode is the positive electrode in a gel tank. The cathode is the negative electrode. DNA carries a negative charge on every phosphate, so the field pulls it toward the anode. Put the wells at the cathode end. If you pour them at the other end, the nucleic acid enters the buffer instead of the gel.

The same direction holds for proteins coated with SDS, which are pulled negative regardless of the protein's own chemistry. A western blot starts from that direction of run, then a second field moves the proteins onto a membrane. Native electrophoresis is the exception to memorise. Without SDS, a protein's net charge can be positive or negative at the buffer pH you chose, and it will run toward the electrode of opposite sign. A native lane read with DNA habits will be backwards half the time.

Leads are labelled by convention, commonly red for the anode and black for the cathode. Confirm with the dye, not with memory, after any repair.

Sieving and mobility

Sieving is what the matrix adds. In free solution a DNA fragment's charge and its friction both grow with length, so different lengths move similarly. In agarose or polyacrylamide the mesh hits long molecules more often than short ones, and length becomes visible as distance. A tighter mesh, from a higher percentage, sieves more strongly and separates smaller species. A loose mesh lets large DNA move and lets small DNA run as a pack.

Very large DNA can move by threading end-on through the mesh. Once that mode dominates, extra length changes the speed less, and the gel stops resolving. That is why a percentage has a window, and why a ladder that flanks the size you care about is part of the claim.

Mobility is speed scaled by the field. A molecule that moves farther in the same time, in the same voltage per centimetre, has the higher mobility. Charge, size, shape, and the mesh all sit inside that number. Supercoiled plasmid and linear plasmid of one sequence have different mobilities. Apparent mass on an SDS gel is a mobility converted by a marker, not a weighing.

Stacking and resolving

In a discontinuous SDS system the gel is poured as two layers. The stacking layer is a low-percentage gel at a pH where the trailing ion, often glycine, moves slowly. The leading ion, often chloride, moves quickly. Proteins gather in a thin zone between those ions and enter the second layer as a narrow band. That gathering is stacking. It is why a sloppy load can still become a sharp band.

The resolving layer is the sieving gel at a higher pH and usually a higher percentage. There the trailing ion speeds up, the stack loosens, and proteins separate by how the mesh retards them. A band's position is decided in the resolving gel. A band that never left the stack, sitting in the well or at the boundary, was too large for that percentage or never fully unfolded. Staining the boundary tells you the failure is entry, not absence.

A western blot inherits both layers. Protein that did not enter the resolving gel will not be a neat band on the membrane. The transfer does not restack it.

Electroendosmosis, briefly

Agarose is not electrically blank. Residual sulfate and pyruvate groups are fixed negatives in the polysaccharide. They attract positive ions from the buffer. When the field is on, those positive ions drift toward the cathode and drag water with them. That flow is electroendosmosis, the same phenomenon instrument notes often call electroosmotic flow. It points opposite to DNA, which is heading for the anode, so it slows the nucleic acid and can blur or distort bands when the agarose is high in those charged groups.

Molecular biology agarose is sold as a low-electroendosmosis grade for that reason. Older agarose meant for serum-protein work can be a poor DNA matrix. The grade belongs in the method file next to the percentage. This is a materials fact, not a reason to add a charged coating of your own.

Rf and the leading dye

The leading dye is the visible tracking dye that travels at or near the ion front. Bromophenol blue is the usual one. It is "leading" because it marks the front that runs ahead of the molecules you care about, not because it is a standard. Xylene cyanol is a second tracking dye that lags behind and is useful when the leading dye would run off before a large fragment has separated. Neither dye has a mass. Their positions change with percentage and buffer. A loading dye's other job is density, so the sample sinks into the well. Density is not electrophoresis.

Rf, sometimes written in full as relative mobility, is a ratio of distances on one gel. Measure from the well, or from a line you state, to the band, and divide by the distance to the front you chose. Use the same front for the marker and the sample. A plot of log mass against Rf is roughly linear in a limited window of the resolving gel. Read inside that window. A western blot image rarely preserves a dye front, so Rf on a blot is a weaker habit than Rf on the stained gel. Prefer the marker bands that actually transferred.

Using the words on a real lane

A straight sentence is: the sample was loaded at the cathode, ran toward the anode through a resolving gel of a stated percentage, passed a leading dye that is still on the gel, and sits between two marker bands. That sentence can support an apparent size. A sentence that quotes the leading dye as if it were 10 kilodaltons, or that reports a mass beyond the marker, cannot.

If the dye never left the well, you do not yet have mobility to discuss. Check the leads and the current before you discuss biology. If the dye front ran off, small species may have gone with it, and Rf has lost its denominator.

Safety and the shared tank

The field is a shock hazard. Covers stay on while current is enabled. Ultraviolet reading of a DNA gel stays behind the orange shield in the photograph. Acrylamide monomer, if you cast the resolving gel yourself, is a separate chemical hazard under the institutional SOP. This glossary does not assign a biosafety level. The sample does.

In a shared room the failure mode is a lead that was resoldered to the wrong colour, or a tank whose cathode end is not the end everyone assumes. Run the leading dye for a minute on a new or repaired tank before you load the only aliquot. Heat that smiles a gel is a mobility change, not a new term. Note the voltage with the distances.

What to ask for

EVRINTH can take a sourcing question that uses these words precisely. State anode-to-cathode distance if you are asking about a tank, the matrix and percentage, whether you need a discontinuous system with a stacking layer, and whether the marker must suit an agarose gel or an SDS gel that may go on to a western blot. The molecular biology catalogue and the quote request are the path. The nucleic acid analysis pathway is the context when the gel is one step in a nucleic-acid workflow. Ask whether a quotation is possible. A product named only "ladder" is not yet a specification.