Affinity Chromatography - Department of Molecular and Cellular ...

Appendix 8
Analytical assays during purification
Analytical assays are essential to follow the progress of purification. They are used to assess
the effectiveness of each step in terms of yield, biological activity, recovery and to help during
optimization of experimental conditions. The importance of a reliable assay for the target
molecule cannot be over-emphasized.
When testing chromatographic fractions, ensure that the buffers used for purification do
not interfere with the assay.
Total protein determination
Lowry or Bradford assays are used most frequently to determine the total protein content.
The Bradford assay is particularly suited to samples where there is a high lipid content that
may interfere with the Lowry assay.
Purity determination
Purity is most often estimated by SDS-PAGE. Alternatively, isoelectric focusing, capillary
electrophoresis, reversed phase chromatography or mass spectrometry may be used.
SDS-PAGE Analysis
Reagents Required
6X SDS loading buffer: 0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol
(or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -80 °C.
1. Add 2 µl of 6X SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates or purified
fractions as appropriate.
2. Vortex briefly and heat for 5 minutes at +90 to +100 °C.
3. Load the samples onto an SDS-polyacrylamide gel.
4. Run the gel and stain with Coomassie Blue (Coomassie Blue R Tablets) or silver (PlusOne Silver Staining Kit,
Protein).
The percentage of acrylamide in the SDS-gel should be selected according to the expected
molecular weight of the protein of interest (see Table 19).
Table 19.
% Acrylamide in resolving gel Separation size range
Single percentage:
5% 36 000–200 000
7.5% 24 000–200 000
10% 14 000–200 000
12.5% 14 000–100 000
15% 14 000–60 000 1
Gradient:
5–15% 14 000–200 000
5–20% 10 000–200 000
10–20% 10 000–150 000
1 The larger proteins fail to move significantly into the gel.
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