Question 5: How can I activate Rho, Rac or Cdc42 GTPases?
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:
T=0 days: Plate cells to 3 to 5% confluency (1 to 3x104 cells/ml).
T=3 days: At ~30% confluency, exchange media to 0.5% serum for 24 h
T=4 days: At ~50% confluency exchange media to 0% serum for overnight (10 to 16 h)
T=5 days: Perform the experiment and freeze the lysates for G-LISA later.
T=5 days: Perform protein assay, calculate dilutions necessary and perform G-LISA.
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 that have 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.
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.
Two usual causes:
- Not flicking and tapping the plate vigorously enough between washes.
Check to make sure that there are no bubbles in the wells. Bubbles will affect the readings.
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. Alternatively, many luminometers have a dynamic range for sensitivity (gain) and integration time. These parameters should be adjusted and evaluated under control conditions to optimize signal detection. It is also important to note that baseline luminescence readings will vary from machine to machine. If the constitutively-active GTPase control protein samples are 3-5 fold higher than lysis buffer blank, the machine is accurately measuring active GTPase levels.
Since Rho and Rho signaling pathways are highly conserved between species, the G-LISA kits are 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/tissue types compatible with G-LISA assays
We recommend preparing positive control sample lysates that can be used with each G-LISA assay. For activation of the Rho GTPase, we recommend treating serum-starved cells for 10-30 min with 0.25-1 unit/ml of calpeptin (cat# CN01). For activation of the Rac GTPase, treatment of serum-starved cells for 2 min with 10 ng/ml of epidermal growth factor is recommended (cat# CN02). For activation of the Cdc42 GTPase, we recommend treatment of serum-starved cells for 1 min with 100 ng/ml of epidermal growth factor (cat# CN02). Additionally, we recommend omission of the PBS wash before lysing cells that have activated Cdc42 as the PBS wash alone can activate this GTPase. Finally, please see the attached table 2 for a list of common activators accompanied by the relevant citation and additional experimental information.
Table 2. Activators of Rho, Rac and Cdc42
We recommend preparing two control cell extracts that can be used to set up the G-LISA assay with your cell or tissue type. A simple activated/control state pair of extracts can be made by growing cells to 50% confluency in serum-containing media, washing twice with room temperature PBS, preparing lysate as normal, aliquoting and freezing in liquid nitrogen. Defrost one aliquot and let stand at RT for 1h which will degrade the signal to a low signal and will be the control state. A second defrosted aliquot to be used immediately will be considered “activated”, which most serum grown cells are.
The Rac and RhoA G-LISA kits use the same lysis buffer. The Cdc42 G-LISA kit (cat# BK127) uses a different lysis buffer. The buffer components are proprietary, but in general, the lysis buffers contain a buffer, detergents and salts. The Cdc42 lysis buffer is about 2X more concentrated than the other lysis buffer in regard to salt and detergent concentrations. So you could make the extracts in the Cdc42 lysis buffer and dilute them in the other lysis buffer for the Rac1 and RhoA assays. As a reminder, be sure to aim for approximately 0.5 - 1 mg/ml protein concentration when performing the lysis. At higher concentrations, you are likely to have significant loss of signal due to proteolysis, increased phosphatase/kinase activity and increased GAP activity.
Yes, tissue lysates can be used instead of cell lysates and the general G-LISA instructions should be followed with the following modification. We recommend isolating the tissue, chopping the tissue into 1 mm chunks and macerating as quickly as possible, perhaps as quickly as 2 min total time. The small GTPase signaling pathway is very quick to change and activated GTPases can be hydrolyzed very quickly. Homogenization can be achieved in the lysis buffer with a micro-pestel and mortar (see Fisher or VWR). Alternatively, 1 mm chunks can be drop frozen in liquid nitrogen for later processing. After homogenization, centrifugation is performed to remove cell debri and the protein concentration is determined. Phosphatase inhibitors are necessary for measuring Rho-GTP in artery samples, but we do not know whether other tissues require these. We recommend the following mixture, a combination of false substrates and inhibitors of serine/threonine phosphatases.
NaF, Final 50 mM, 100 µl of 500 mM stock
Na pyrophosphate, Final 20 mM, 100 µl of 200 mM stock
p-Nitrophenyl phosphate, Final 1mM, 100 µl of 10 mM stock
Microcystin LR, Final 1 µM, 2.5 µl of 400 µM stock (in 10% ethanol)
Cytoskeleton Lysis buffer, 700 µl
Cytoskeleton protease inhibitors (Cat. # PIC02), 10 µl
There are several things to consider when dealing with potential variation in the experimental tissue samples, e.g., GAP activity, GEF activity, phosphatase activity, physical treatment and time to freezing. To reduce the enzyme activities, you can prepare the lysates as described above.
A good test for lysates is to see what happens to them when you incubate at RT for 0, 2, 10 and 20 min. If the signal goes down rapidly, then your extracts need stabilizing. Try to aim for an extract concentration of 0.5 mg/ml because then the enzyme activities will be reduced.
High activation states under non-stimulated conditions might be due to the GEF activity or the physical treatment during excision (try a new sharp scalpel and slice rather than chop the tissue). 1 mm3 chunks should be frozen in liquid nitrogen. If these suggestions do not help, then try adding 300 mM NaCl and 0.2% SDS to the lysis buffer as this will reduce the GEF and GAP activities by reducing their interaction with the small G-proteins.
Cells grown in 3-D culture in Matrigel or collagen gels can be assayed using the luminescence-based (BK121 and BK126) kits. The low amounts of cells in 3D cultures makes the conventional pull-down assay very difficult, variable and expensive. Customers have successfully used the G-LISA assay with cells grown in these conditions. Please see Keely et al., 2007 (Methods in Enzymology, v426, p27) and Petroll et al., 2008 (J Cell Physiol, v217, p162) for in-depth descriptions using G-LISA technology and 3-D cultures. An additional modification we suggest is to increase the ratio of extract/binding buffer from the recommended 1:1 (v/v) to 1:3.
The RhoA G-LISA kit (Cat. # BK121 and BK124) can be used to detect RhoB or RhoC. The Rac G-LISA (Cat. # BK125 and BK128) can be used to detect Rac2 or Rac3. The RalA G-LISA (Cat. # BK129) can be used to detect RalB. In all cases, the G-LISA wells capture multiple isoforms of the respective GTPase and the specificity of the primary antibody added to the wells determines which activated isoforms are detected and quantified. We recommend evaluating antibodies by western blotting of purified isotype-specific G-proteins and then titrating these verified primary antibodies with the respective G-LISA kit. Please see Hall et al., 2008 (Neoplasia. v10, p797) for a successful example of this modification of G-LISA technology. Recently, a customer has also reported that a RhoB antibody available from Santa Cruz worked quite well with our RhoA G-LISA kit.
At Cytoskeleton, Inc., we use titer plate shakers (model# 4625) from Lab-Line Instruments. These models are also compatible:
Model # RF7854 Digital Microplate Shaker, ML Market Lab, researchml.com
Model # RF7855 Incubating Microplate Shaker, ML Market Lab, researchml.com
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