Categorized: Biochemical Assays
Wright and Rebers, Anal. Biochem. 49: 307-319, 1972.
To determine the relative amounts of LPS carbohydrates present in a given strain. The assay can be done on one set of samples and then scanned at the various wavelengths for reasonable data on the 3 types of sugars.
(sugars in the outer core and O-Ag)
- Cool 200 µl of sample in an ice bath.
- Add 900 µl reagent A (4 ml dH20 + 24 ml conc H2SO4). Shake carefully. Warm to room temperature (23ºC).
- Boil in water bath for exactly 20 min.
- Cool to room temperature under cold water tap.
- Add 20 µl reagent B ( 0.3 g cysteine HCl in 10 mls dH2O (fresh)). Shake and then let stand 30 min. in the dark at room temperature.
- Assay the difference between 415 and 380 nm.
- To zero the spec use all above ingredients except the bacterial sample. Standards are made of 1,2,3,4, and 5 µl of desired hexose sugar (eg. glucose) at 2 µg/ml.
- To assay all three kinds of sugars do a wave length scan from 350 – 600 nm.
6-Deoxyhexose Assay: (outer core only)
Examples are fucose and rhamnose. The assay is the same for the hexoses but it is read at 396 and 427 nm. To make this assay more specific for 6-deoxyhexoses use the following modifications:
- In step 3 above, boil for 5 min.
- In step 6 above, let stand for 3 hours in the dark (this produces a green-yellow colour rather than pink)
- Standards are the same as above using rhamnose.
Heptose Assay: (inner core only)
Same as above but uses the difference between 500 and 545 nm.