Q: I am concerned about being able to detect low levels of active small GTPase in my sample using your Small GTPase Activation Assays. Do you have any suggestions to ensure I get good results?
A: There are two things you can do to improve your results with our Small GTPase Activation Assays:
- Before performing the pull-down, it is always a good idea to first ensure you are able to detect your target of interest. This may be done by performing a Western blot directly on your lysate using the antibody supplied in the kit, without performing the pull-down.
- When performing the pull-down, use as much lysate as possible. Since the active form is usually a very small percentage of the total, it is recommended to use 100 times the lysate amount needed to see a clearly defined band in the aforementioned direct Western blot.
Q: I am studying the role of an active Ras family member. Can I use conventional Western Blotting instead of using your Small GTPase Activation Assays to detect the active form?
A: Strictly relying on Western blot for detection of any active small GTPase is not recommended. We are not aware of any antibody that will reliably bind only to the GTP (active) form and not to the GDP (inactive) form.
Q: Are the Small GTPase Assay Beads (Raf-RBD, PAK1-PBD and Rhotekin-RBD) species specific?
A: Sequence alignment of a specific small GTPase indicates that there is at most one or two amino acid variation between various species. Therefore our beads and also our primary antibodies may be used across many species.
Q: Can these assays be used with tissue lysate samples?
A: Yes, our small GTPase activation assays are compatible with tissue lysate samples. Tissue samples can be resuspended at 20-50mg/ml in 1X Assay/Lysis buffer or other lysis buffer containing proteinase inhibitors. After homogenization, spin and collect the supernatant as the lysate.
Q: Can I use a different lysis buffer?
A: It is fine to use a different lysis buffer, such as RIPA buffer. The recipe of the 5X Assay/Lysis buffer is provided here if you need to make more: 5X Assay/Lysis Buffer: 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% NP-40, 50 mM MgCl2, 5 mM EDTA, 10% Glycerol.
Q: Do the controls need to be run with each sample, or just once for the assay?
A: The GDP and GTPgS loading controls are assay controls to verify that the pull down was performed correctly. Since the controls are independent of the endogenous small GTPase activity of your samples, they only need to be run once.