9780521516358book.pdf

relative molecular mass of the protein can be determined on the same gel run

(as described above), with no more material being used.

10.3.2 Native (buffer) gels

While SDS–PAGE is the most frequently used gel system for studying proteins, the

method is of no use if one is aiming to detect a particular protein (often an enzyme) on

the basis of its biological activity, because the protein (enzyme) is denatured by the

SDS–PAGE procedure. In this case it is necessary to use non-denaturing conditions. In

native or buffer gels, polyacrylamide gels are again used (normally a 7.5% gel) but the

SDS is absent and the proteins arenotdenatured prior to loading. Since all the

proteins in the sample being analysed carry their native charge at the pH of the gel

(normally pH 8.7), proteins separate according to their different electrophoretic mobi-

litiesandthe sieving effects of the gel. It is therefore not possible to predict the

behaviour of a given protein in a buffer gel but, because of the range of different

Example 1MOLECULAR MASS DETERMINATION BY ELECTROPHORESIS

Question The following table shows the distance moved in an SDS–polyacrylamide gel by a
series of marker proteins of known relative molecular mass (Mr). A newly purified
protein (X) run on the same gel showed a single band that had moved a distance of
45 mm. What was theMrof protein X?

Protein Mr Distance moved (mm)

Transferrin 78 000 6.0

Bovine serum albumin 66 000 12.5

Ovalbumin (egg albumin) 45 000 32.0

Glyceraldehyde-3-phosphate dehydrogenase 36 000 38.0

Carbonic anhydrase 29 000 50.0

Trypsinogen 24 000 54.0

Soyabean trypsin inhibitor 20 100 61.0

b-Lactoglobulin 18 400a 69.0

Myoglobin 17 800 69.0

Lysozyme 14 300 79.0

Cytochromec 12 400 86.5

Note:ab-lactoglobulin has a relative molecular mass of 36 800 but is a dimer of two identical

subunits of 18 400 relative molecular mass. Under the reducing conditions of the sample buffer

the disulphide bridges linking the subunits are reduced and thus the monomer chains are seen

on the gel.

Answer Construct a calibration graph by plotting logMrversus distance moved for each of the

marker proteins. From a graph of logMrversus the distance moved by each protein

you can determine a relative molecular mass for protein X of approximately 31 000.

Note that this method is accurate to10%, so your answer is 31 0003100.

410 Electrophoretic techniques