Figure 11.13 Electrophoresis of human serum proteins. The electrophoretogram, after staining with a suitable dye, can be scanned using a densitometer, which gives a trace of the absorbance pattern of the strip.
> Reflectance - see Section 2.3.5.
some types of cellulose acetate material, using an ethanol-acetic acid-ethylene-glycol reagent, which causes the collapse of the porous structure of the membrane rendering it transparent. Some densitometers are designed to measure reflected light rather than transmitted light and so enable the use of opaque strips. This simplifies the technique but does introduce a further factor in the relationship between absorbance and the amount of protein present in the sample. This relationship is still valid for reflectance measurements provided that the amount of protein involved is very small, and hence such instruments are usually designed for micro-analytical techniques.
Procedure 11.7: Location of proteins after electrophoresis on cellulose acetate llliM
Trichloroacetic acid 75.0 g 1 !
Immerse the electrophoretogr.im in the stain tor 5 to 10 min.
Remove and clear the background of excess stain h\ soaking in dilute acetic acid (50 g I '). Measure the absorh:uice at 520 nm using a densitometer.
> Polyacrylamide gel electrophoresis - see Section 3.3.2.
In the capillary electrophoresis of proteins it is essential that the buffer and the medium have good transparency in the ultraviolet to enable effective detection after the separation. Polyacrylamide gel does present a problem in this respect, interfering with protein detection at 214 nm. As a consequence of the commercial impact on capillary electrophoresis, a range of ready-to-use media is available from manufacturers.
Proteins can be dissociated into their constituent polypeptide chains by the detergent sodium dodecyl sulphate (SDS) after the reduction of any disulphide bonds. The SDS binds to the polypeptide chain producing a rod-shaped complex, the length of which is dependent upon the relative molecular mass of the protein. The large number of these strongly anionic detergent molecules bound by the protein (approximately equal to half the number of amino acid residues) effectively masks the native charge of the protein and at a neutral pH results in a relatively constant charge to mass ratio for all proteins. As a result, the elec-trophoretic mobility of all protein-SDS complexes is approximately equal but the molecular sieving effect of polyacrylamide gel results in a relative mobility which is inversely related to the size of the complex. Under certain conditions, this inverse relationship can be demonstrated by a linear plot of the relative mobility of the protein against the logarithm of its relative molecular mass (Figure 11.14). It is necessary to use a series of known proteins in order to produce a calibration curve and kits are available commercially for this purpose.
Prior to electrophoresis the sample is diluted in buffer containing SDS (10-25 g l"1) and /3-mercaptoethanol (10-50 ml l"1), which reduces any disulphide bonds stabilizing the protein. It is then heated at 100 °C for 2-5 min in order to denature the protein and expose the total length of the polypeptide chain to the detergent. After cooling, electrophoresis is performed on Polyacrylamide gel and the bands subsequently visualized using an appropriate dye.
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