Choosing an activation assay
Step 1: Choose the number of assays required.
The number of assays can be as little as 10 to 20 for a quick look & see experiment, for a single substance or gene, or upto a couple of hundred assays if there are many conditions to study. Even in the simplest case the timing and extent of activation can change considerably, see Figure 1. For example LPA has a very short transient activation (less than 3min), whereas calpeptin is longer and more stable (10-30 min) and CNF (Cat. CN03) takes longer still and creates a very large signal. It is clear that we should be aware of the need to use multiple time points even in the quickest look & see analysis.
 |
Figure 1. Time course of RhoA activation by 10 µg/ml LPA, 100 µg/ml calpeptin (Cat.# CN01) and 1 µg/ml CNF (Cat.# CN03) in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with activator for the times shown in the graph and RhoA activation was analyzed with G-LISA kit BK124. LPA stimulation leads to a rapid and transient activation of RhoA, which peaks at 2-3 min and quickly declines to basal levels. Activation by calpeptin leads to a signal increase between 10-30 min which declines to basal after 60 min. Activation by CNF leads to signal increase after 60 min which is stable for up to 12 h. |
Choose the number of samples you require.
