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Study Notes: Electrophoresis Buffers

A simple buffered solution contains a mixture of a weak acid (HA) and its conjugate (A-) base according to the following equation:

HA equilibrium symbol H+ + A-

The position of this acid/base equilibrium (that is whether it lies well to the left or right of the equation) is represented by the acid dissociation constant Ka.

The number is large if the acid is stronger and equilibrium tends towards dissociation (the right of the equation). If it is small the acid is weaker and equilibrium tends towards proton (H+) capture (the left of the equation).

Buffers used in the life sciences generally have Ka values ranging from 10-4 to 10-10.

K to the power of a equals H positive multiplied by A negative divided by HA

Ka is usually expressed as its negative logarithm pKa:

pKa = - log Ka

That is, a buffer with a Ka of 10-2 has a pKa of 2, favouring dissociation (acid), whilst a buffer with a Ka of 10-12 has a pKa of 12, favouring proton capture (basic).

The equation for the dissociation constant can be converted into the very useful Henderson-Hasselbalch equation:

pH equals pK to the power of a plus log basic form divided by acidic form

This equation is fundamental to buffer formulation and shows that the pH of a buffered solution will differ from the buffer’s pKa by an amount determined by the ratio of the base to the acid form in solution.

If these concentrations are equal then pH = pKa.
If the concentration of the base form is greater than the acid then the pH > pKa.
If the concentration of the acid form is greater than the base then pH < pKa.

A buffer maintains a pH close to constant by absorbing protons released from other sources in solution or releasing protons if another species is depleting them.

For example, a small amount of strong base added to pure water causes the pH to move from 7 (neutral) to 12-13 (basic).

But if the same amount of strong base is added to a concentrated buffer solution it merely causes some of the weak base form to change to the weak acid form. This will counteract the effect of the added base with the result that the pH would barely alter or not change at all.

Under the conditions of electrophoresis, the buffers used are able to maintain a relatively constant pH as long as either the acids or base forms do not become exhausted. The buffer system in electrophoresis controls the pH of the gel, preventing damage to sample molecules and sometimes also controlling the ionisation of the sample molecules.

The buffer also provides the ions for the vast majority of current in the system.

  • In denaturing PAGE electrophoresis of DNA (where top and bottom tank buffers are the same), the buffer prevents large changes in pH and controls the conductivity of the gel.
  • In native electrophoresis of proteins, the buffer pH also controls the ionisation state of the samples.
  • In a discontinuous multiphasic system such as SDS-PAGE (different pH buffers in the gel and tanks) buffer design and use becomes even more complex.

Some buffer points:

  • In all cases, the ionic strength of the buffer must be sufficient to keep the sample in solution in the gel.
  • Higher concentrations of gel buffer slows diffusion of bands and produces sharper bands.
  • Higher buffer concentrations allow higher conductivity leading to possible temperature problems. To overcome this problem high buffer concentrations are used in conjunction with constant voltage.

Some further buffer points:

  • The buffer should be chosen with a pKa that is very close to the desired pH - within half a pH point.
  • Buffers that form ions with high charge (+2, +3, -2 etc.) are hard to work with as this high charge increases current and depletes the ions in the buffer rapidly. Tris base and borate make excellent buffer species as they are uncharged for part of the time at the desired pH (that is, will have a reduced electrophoretic mobility).
  • Buffer species with high molecular size also move slowly in electrophoresis. For example, Tris base moves more slowly than ions such as chloride or phosphate because of its low charge/mass ratio and this property inhibits buffer depletion.

Commonly used buffers are listed in the following table. Note the differences in molecular weight and pKa.

Buffer Molecular Weight (Mr) pKa
Acetic Acid 60.05 4.8
Boric Acid 61.68 9.23
Glycine 75.07 9.8
Tricine 179.18 8.15
Tris 121.1 8.06

A few words about buffer systems. There are a number of different buffer configurations that are used for different kinds of electrophoresis.

Homogenous Buffer System - the identity and concentration of buffer components are the same in the gel and both tanks. This is used for most forms of DNA and RNA electrophoresis.

Multiphasic Buffer Systems - as the name implies this system uses differing buffers and is used for SDS-PAGE (often called the Laemmli system). The Laemmli system uses an additional gel layer above the separating gel. This is a lower percentage acrylamide gel with a different buffer. This gel acts to ‘stack’ the protein bands into sharp zones prior to separation in the higher strength separating gel. Buffers used are:

  • Stacking Gel - Tris-HCl, pH 6.8
  • Separating Gel - Tris-HCl, pH 8.8 at a higher concentration
  • Tanks - Tris-glycine, pH 8.8.

Isotachophoresis - uses a non-sieving low percentage gel (essentially one large stacking gel) and is used for difficult samples such as very small peptides (small proteins).

Buffer Additives - are usually added to the buffer perfusing the gel and can include:

  • Hydrogen bonding agents such as urea and formamide, which disrupt hydrogen bonds, that affect the conformation and solubility of biological molecules.
  • Surfactants such as Triton X-100, Tween 20 or SDS. SDS is the most commonly used detergent. This causes the protein chains to unwind from their native configuration and the protein is said to be denatured.
  • Reducing Agents - such as 2-mercaptoethanol or dithiothreitol that break the disulphide bond linkages that hold protein chains together. The protein is then said to be reduced. Usually added to the sample rather than the gel as they inhibit formation of the gel.

This image shows how the detergent SDS attaches to the protein and imparts a negative charge to the protein.
Surfactant molecules attached to protein backbone

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