Estimating molecular weight by polyacrylamide gel electrophoresis

B.2 Estimating molecular weight by polyacrylamide gel electrophoresis

B.2.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Electrophoresis involves the migration of proteins and is dependent upon the charge, size and shape of the molecules. With SDS-PAGE, proteins bind the SDS and become negatively charged, so have similar charge : weight ratios. When SDS-coated proteins are placed in an electric field, their spatial separation will depend only upon their size and shape. By varying the concentration of the polyacrylamide gel used as the medium for the electrophoretic separation, different resolu- tion ranges of molecular weights may be obtained. Proteins may be fractionated in the native state, but better resolution is usually obtained if the disulphide bonds are first reduced, allowing separation of the individual peptide chains.

Briefly, the technique involves the protein solution being heated to 100°C in the presence of reducing agents and SDS; the proteins unfold and bind about 1.4 g SDS/g protein. The strong negative charge on the proteins means that their electrophoretic mobility is inversely propor- tional to the logarithm of their molecular weight. There are some exceptions to this behaviour and they include: (a) heavily glycosylated proteins, which bind less SDS than unglycosylated molecules of similar

molecular weight; and (b) some proteins, e.g. immunoglobulin J chains, which do not unfold completely and retain some of their native configuration.

A stacking gel is cast on top of the separating gel with a lower percentage polyacrylamide con- centration (typically between 3 and 5%) and is prepared using a buffer with a slightly different composition. The different mobilities of chloride and glycine and the slow rate of entry into the

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MATERIALS AND EQUIPMENT Vertical slab gel system Acrylamide N,N′-methylene bis acrylamide

3.0 M tris(hydroxymethyl)-aminomethane (Tris)–HCl buffer, pH 8.7

1.0 M Tris–HCl buffer, pH 6.8 Ethylene diamine tetra-acetic acid (EDTA) Sodium dodecyl sulphate (SDS) N,N,N′,N′-tetramethylethylene diamine (TEMED) Water-saturated butan-3-ol Ammonium persulphate Bromophenol blue Sucrose

Separating gels

6% Stacking gel 5%

40% acrylamide

11.25 7.5 5.6 4.5 1.25 1% bis acrylamide

3.75 3.75 3.75 3.75 1.25 10% ammonium persulphate

3 M Tris–HCl, pH 8.7

0.2 0.2 0.2 0.2 0.1 Distilled H 2 O

6.84 10.35 12.25 13.35 5.0 20% SDS

0.15 0.15 0.15 0.15 0.05 TEMED

0.02 0.02 0.02 0.02 0.01 100 m M EDTA

1.0 Molecular weight range

175 000 * Use 1 m Tris–HCl buffer for the stacking gel.

Casting the gel

1 Wash thoroughly plates, spacers, etc., rinse in distilled water and air dry. Rinse in 70% ethanol and dry.

2 Assemble the slab gel mould, check it is vertical.

A P P E N D I X B: Basic techniques and useful data

3 Mix the solutions for the separating gel according to the table above, but omit the SDS and TEMED at this stage. The precise final volume of reagents needed depends on the type of equipment being used, but the table gives a guide to the proportions required to produce gels for separation in various molecular weight ranges.

4 De-gas the solution under vacuum.

5 Add the SDS and TEMED and run the solution between the plates, within 4 cm of the top of the plates. Take care to avoid air bubbles.

6 Overlay the top of the separation gel with aqueous isobutanol (butan-3-ol saturated with distilled water). This reduces surface tension and ensures that the polymerized gel will have a flat surface.

7 Leave to polymerize for about 30–60 min and then wash off the butanol with three changes of distilled water and dry the surface carefully with filter paper. Alternatively, prepare an excess of stacking gel solution and use this to rinse away all traces of the isobutanol.

8 Mix the stacking gel and de-gas under vacuum prior to the addition of TEMED and SDS, pour into the mould over the separating gel and insert the plastic comb to form the sample wells. Leave the stacking gel to polymerize for 30 min.

Sample preparation

MATERIALS Polyacrylamide sample buffer

Dithiothreitol Bromophenol blue Molecular weight standards, e.g. rainbow markers

METHOD

1 If the sample to be analysed is in a strong buffer, dialyse it against sample buffer, otherwise proceed to step 2.

2 Add 40 µl sample (containing 2–20 µg protein) to 20 µl of sample buffer (containing

31 mg/ml dithiothreitol) and 3 µl bromophenol blue (1 mg/ml in water). For unreduced samples omit the dithiothreitol.

3 Prepare the molecular weight standards in the same way.

4 Heat for 3 min in a boiling water bath.

Running the gel

MATERIALS AND EQUIPMENT Power supply Gas-tight glass syringe (50 µl) Plastic capillary tubing Polyacrylamide running buffer: tris(hydroxymethyl)-aminomethane (Tris)–glycine, pH 8.3,

containing 3 ml 20% sodium dodecyl sulphate (SDS) per 600 ml buffer

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METHOD

1 Carefully remove the comb from the polymerized gel, clean wells of any unpolymerized acrylamide with running buffer, mount the gel plate in the electrophoresis apparatus and fill with running buffer.

2 Add samples to wells using a 50-µl syringe with plastic capillary tubing or a fine pipette. Do not forget the molecular weight standards.

3 Connect to power supply with anode at the bottom. Run at constant current: 20 mA while the sample is in the stacking gel and 40 mA after it enters the separating gel. The precise power requirements will vary according to the length and thickness of gel and whether the apparatus has a cooling pattern.

4 Once the bromophenol blue marker dye is approximately 1 cm from the bottom of the gel, turn off the power and remove the gel for either fixation and staining or electrophoretic transfer to nitrocellulose (see Chapter 4).

TECHNICAL NOTES • Although this is a relatively robust technique, it is prone to artefacts from various sources. If too

much current is applied to speed separation, then heating effects can distort the separation pattern and are seen as a horizontal wave pattern after protein staining. Either use cooling or reduce the current across the gel. Individual tracks are sometimes seen to give vertical streaks; this is due to a high salt content in the initial sample allowing a local increase in the current. This may be corrected by sample dialysis against the running buffer.

• Samples in the outside tracks may show an upward curve in their polypeptide bands due to greater electrical resistance at the edge of the gel. This is usually only a problem if the gel is being run to analyse and compare complex protein mixtures. This may be eliminated by running a sample of an irrelevant protein, such as bovine serum albumin, in the outside tracks.

• It is possible to prepare an ‘in-house’ mixture of molecular weight standards by buying pure proteins from one of the chemical suppliers. In addition, these can be radiolabelled using 14 C- formaldehyde. However, commercial mixtures are now available which contain intensely dyed proteins. It is therefore possible to monitor the progress of polypeptide bands across the whole gel during electrophoresis.

B.2.2 Staining and molecular weight estimation

To locate and recover a particular polypeptide band, e.g. for use in T-lymphocyte stimulation assays, we recommend the use of Aurodye rather than the methods described below. This pro- cedure is said to be as sensitive as silver staining and yet does not affect mitogenesis assays.

Proteins (minimum detection limit 1 µg)

MATERIALS Methanol Acetic acid Coomassie blue

A P P E N D I X B: Basic techniques and useful data

METHOD

1 Fix the gel in a mixture of 40% methanol, 10% acetic acid for 4 h.

2 Stain in 0.1% w/v Coomassie blue in methanol–acetic acid for 5 h.

3 Destain in 30% methanol, 10% acetic acid for 2 h.

4 Complete the destaining in 10% methanol, 10% acetic acid.

5 Reswell and store in 7% acetic acid.

Carbohydrates (minimum detection limit 5 µg)

MATERIALS Methanol Acetic acid Periodic acid Schiff’s reagent Sodium metabisulphite

METHOD

1 Fix gel in 40% methanol, 20% acetic acid for 4 h.

2 Reswell in 7% acetic acid.

3 Oxidize in a mixture of 1% periodic acid in 7% acetic acid for 1 h in the dark.

4 Wash in 7% acetic acid for 24 h, changing the wash several times.

5 Stain with Schiff’s reagent at 4°C for 1 h in the dark.

6 Differentiate in 1% w/v sodium metabisulphite in 0.1 M hydrochloric acid. The apparent relative molecular weight (app. MW r ) of unknown polypeptide or glycopeptide

bands may be determined by reference to the set of internal standards which should be run in each gel.

1 Identify each polypeptide band in the track of molecular weight standards and measure its migration distance from the interface between the stacking and separating gels.

2 Plot a graph of log molecular weight against distance travelled and use this to read back from the distance travelled by the unknown band to its log molecular weight.

TECHNICAL NOTES • The molecular mass estimate determined by this technique is an apparent relative molecular

weight as it is obtained by comparison with the set of molecular weight marker proteins run in the same gel. The value thus obtained should be expressed as a simple number, without units.

• Size determination by this technique can produce surprising and dramatic deviations from

reality, due to artefacts produced by unexpected behaviour or unrecognized peculiarities of the unknown protein, for example: (a) Unfolding of a protein mixture to random coils may occur to different degrees. (b) Inactivation of proteinases from cell-based assays may be incomplete, thus permitting pro-

tein degradation during sample preparation. Some proteinases are poorly inactivated by boiling and SDS treatment; consequently, the unfolded proteins are likely to be even more susceptible to proteolytic cleavage than in their native state.

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(c) The unknown proteins might be heavily glycosylated or phosphorylated and so not show the full charge for weight gain expected after SDS treatment. When some glycophospho- proteins have been compared as native proteins and in vitro translation products by this technique, the latter show increased app. MW r , even though the molecular mass of the unglycosylated and unphosphorylated in vitro-derived protein is smaller.

• A reducing agent, dithiothreitol or 2-mercaptoethanol, is included to reduce both inter- and intrachain disulphide bonds. Remember to include unreduced samples when analysing unknown proteins by this technique.

B.2.3 Gradient gels

Homogeneous polyacrylamide gels are widely used, partly because of the ease of pouring these gels, but gradient gels give increased resolution. Gradients of varying ranges may be prepared, e.g. from 4 to 30%. Proteins continue to move within the gel until they effectively reach their pore size in the sieving gradient.

B.2.4 Two-dimensional SDS-PAGE

Increased information may be obtained about a protein by running a two-dimensional electrophoresis.

A conventional SDS-PAGE may be run in one direction in non-reducing conditions, then fol- lowing incubation of the gel with reducing agent, the proteins may be run into a second reducing SDS-PAGE. From this analysis the molecular weight of the native protein may be calculated and also, if the protein is made up of several polypeptide chains, this will be revealed in the second dimension.

Alternatively: the first dimension may be run as an isoelectric focusing gel, to give information on the isoelectric point, and the second dimension can be run as an SDS-PAGE to reveal the molecular weight.