Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity.
Vector Labs offers a series of sugars that are intended for such a purpose.
The lectin is diluted to a suitable working concentration in a solution containing approximately
200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min. Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section (membrane blot, etc.) will be eliminated. Some trace binding to the section (blot etc) may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding