This page will be continously updated with technical tips for the G-LISA™ assays based on feedback we get from customers.
If you have bought a G-LISA™ kit and have comments, your own technical tips or want to tell us how it worked for you, we encourage you to contact us. Click here to submit feedback.
High variability between samples when using absorbance based G-LISA™ (Cat. # BK124, BK125 and BK127)
Will the G-LISA™ assay work for my specific cell type?
My signals are weak and I sometimes have problems with high variability between samples
Can G-LISA™ be used for cells grown in matrigel or other 3-D growth conditions?
| Issue |
Possible solution |
|||||||||||||||||||||||||||||
| 1. The differences in Rho activation between control samples and stimulated samples are smaller than expected. |
Three different solutions to this problem may exist:
A: If your control cells are supposed to be serum starved, the lack in difference could be due to inefficient serum starvation, i.e. high basal Rho activation levels. To achieve good serum starvation and inactivation of Rho with most cells, it is important to have had the cells growing in the plates for at least 3 days before the starvation is started. We recommend the following protocol for serum starvation:
B: You could be looking too late or too early to see the maximum effect on Rho activation. We recommend to always do time curves for stimuli with unclear effects on Rho activation. For example, LPA is a well-known Rho activator that has Rho-dependent effects on cells and their cytoskeleton for prolonged times. The Rho activation seen by LPA treatment, on the other hand, is very transient. The spike in measurable Rho activity is often gone within 10 min (see Fig 3 on the main G-LISA™ page). C: Too much time may have elapsed between the cell lysis and the addition of the concentration-equalized lysates to the G-LISA™ wells. This will lead to smaller differences between samples. One way to get around this problem, especially if you are handling large numbers of samples, is to snap-freeze the samples in liquid nitrogen as soon as possible after lysis and clarification. To do this, lyse your cells, clarify the lysates, take off 30 µl of lysate for concentration determination and snap freeze the remaining sample. This way, protein concentrations and dilutions needed can be determined without time pressure. Once all samples are prepared and measured with regards to protein concentration, thaw all samples quickly, dilute as needed and add to the G-LISA™ wells according to the manual. |
|||||||||||||||||||||||||||||
| 2. High variability between samples when using colorimetric based G-LISA™ (Cat. # BK124, BK125 or BK127 | Two usual caused:
1. Not flicking and tapping the plate vigorously enough between washes. 2.Check to make sure that there are no bubbles in the wells. Bubbles will affect the readings. |
|||||||||||||||||||||||||||||
| 3. Will the G-LISA™ assay work for my specific cell type? | Since Rho and Rho signaling pathways are highly conserved between species, the G-LISA™ kit is likely to work for all mammalian species and many other eukaryotic organisms. We are compiling a list of cell lines and species where G-LISA™ has been tested. See Table 1 at the bottom of this page. If you have used G-LISA™ with a cell type or species not listed in the table, we would very much like to hear from you. Email us about your experience. | |||||||||||||||||||||||||||||
|
Table 1. Examples of cell types that G-LISA™ has been successfully used with
|
||||||||||||||||||||||||||||||
| 4. My signals are weak and I sometimes have problems with high variability between samples | We have recently updated the BK124 kit (version 1.5). This new version of the G-LISA™ kit gives stronger signals (see Fig. 1) and lower variability between samples, allowing you to get more consistent data and/or use even less cell material with confidence. | |||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||
| 5. Using the luminescence based kit (Cat. # BK121), I get too high background readings with my luminometer | Users have successfully used a gel scanner to read the plate and achieve lower readings with a 12:1 ratio between positive control and background readings. | |||||||||||||||||||||||||||||
| 6. Can G-LISA™ be used for cells grown in matrigel or other 3-D growth conditions? | Cells grown in 3-D culture in Matrigel or collagen gels can be assayed using the BK121 kit. The low amounts of cells in these gels makes the conventional pull-down assay very difficult, variable and expensive. Customers have successfully used G-LISA with cells grown in these conditions by increasing the ratio of extract/binding buffer from the recommended 1:1 (v/v) to 1:3 . | |||||||||||||||||||||||||||||
