Question

6. Assume you are given a mixture of proteins that you analyze by standard 2-D electrophoresis, with the following results for isoelectric points and apparent molecular weights: protein A (Mr 45,140; pI 7.52), protein B (Mr 74,400; pI 5.21), and protein C (Mr 108,370; pI 6.26).

a. In the SDS-PAGE step, in what order did the proteins band, starting nearest the anode and going toward the cathode? Explain why.

b. Which protein would be considered the most basic? Explain why.

c. If you subject the mixture of proteins A, B and C to gel filtration on a 100 ml Sephadex G200 column and find that C elutes at 40 ml and A at 80 ml, at what volume will protein B elute?

d. If protein D (Mr 91,385; pI 6.66) were added to the mixture of proteins A, B and C and the new mix subjected to SDS-PAGE, at what position would D be found in the gel relative to proteins B and C? Express you answer as the fractional distance between proteins C and B at which D bands, i.e., CD distance/CB distance.

e. If the mixture of A, B, and C is subjected to salting out with ammonium sulfate at pH 6.5, in what order would the proteins precipitate? Explain why.

f. If the mixture of A, B, and C is subjected to P-cellulose (pKa1 3, pKa2 6) chromatography, what optimal pH range should you use for the buffer and in what order will the proteins emerge from the column? Explain why.

Answer 1

Multiple components of a single sample can be resolved most completely by two-dimensional electrophoresis (2D-PAGE). The first dimension separates proteins according to their native isoelectric point (pI) using a form of electrophoresis called isoelectric focusing (IEF). The second dimension separates by mass using ordinary SDS-PAGE. 2D PAGE provides the highest resolution for protein analysis and is an important technique in proteomic research, where resolution of thousands of proteins on a single gel is sometimes necessary.

To perform IEF, a pH gradient is established in a tube or strip gel using a specially formulated buffer system or ampholyte mixture. Ready-made IEF strip gels (called immoblized pH gradient strips or IPG strips) and required instruments are available from certain manufacturers. During IEF, proteins migrate within the strip to become focused at the pH-points at which their net charges are zero. These are their respective isoelectric points.

The IEF strip is then laid sideways across the top of an ordinary 1D gels, allowing the proteins to be separated in the second dimension according to size.

Overview of 2D gel electrophoresis. In the first dimension (left), one or more samples are resolved by isoelectric focusing (IEF) in separate tube or strip gels. IEF is usually performed using precast immobilized pH-gradient (IPG) strips on a specialized horizontal electrophoresis platform. For the second dimension (right), a gel containing the pI-resolved sample is laid across to top of a slab gel so that the sample can then be further resolved by SDS-PAGE.

In SDS-PAGE, the gel is cast in buffer contain sodium dodecyl sulfate (SDS) and protein samples are heated with SDS before electrophoresis so that the charge-density of all proteins is made roughly equal. Heating in SDS, an anionic detergent, denatures proteins in the sample and binds tightly to the uncoiled molecule. Usually, a reducing agent such as dithiothreitol (DTT) is also added to cleave protein disulfide bonds and ensure that no quaternary or tertiary protein structure remains. Consequently, when these samples are electrophoresed, proteins separate according to mass alone, with very little effect from compositional differences.

When a set of proteins of known molecular weight are run alongside samples in the same gel, they provide a reference by which the mass of sample proteins can be determined. These sets of reference proteins are called molecular weight markers (MW markers) or standards, and they are available commercially in several forms. SDS-PAGE is also used for routine separation and analysis of proteins because of its speed, simplicity and resolving capability.

Protein samples prepared for SDS-PAGE analysis are denatured by heating in the presence of a sample buffer containing 1% SDS with or without a reducing agent such as 20mM DTT, 2-mercaptoethanol (BME) or TCEP. The protein sample is mixed with the sample buffer and boiled for 3 to 5 minutes, then cooled to room temperature before it is pipetted into the sample well of a gel. Loading buffers also contain glycerol so that they are heavier than water and sink neatly to the bottom of the buffer-submerged well when added to a gel.

If a suitable, negatively charged, low-molecular weight dye is also included in the sample buffer, it will migrate at the buffer-front, enabling one to monitor the progress of electrophoresis. The most common tracking dye for sample loading buffers is bromophenol blue. Thermo Scientific Lane Marker Sample Buffers contain a bright pink tracking dye.

Samples may contain substances that interfere with electrophoresis by adversely affecting the migration of protein bands in the gel. Substances such as guanidine hydrochloride and ionic detergents can result in protein bands that appear smeared or wavy. The Thermo Scientific Pierce SDS-PAGE Sample Prep Kit facilitates removal of these interfering components using a specialized affinity resin system. Methods such as this are much faster and than dialysis, ultrafiltration or acetone precipitation and the protein recovery is generally higher.