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: Do you have a detailed protocol for using the beads?
A: The protocol for these beads is similar to any of our Small GTPase Activation Assays. Here is a protocol for the pulldown assay:
- Aliquot 0.5 – 1 mL of cell lysate to a microcentrifuge tube.
- Adjust the volume of each sample to 1 mL with 1X lysis buffer.
- Thoroughly resuspend the agarose bead slurry by vortexing or titurating.
- Quickly add 40 µL of resuspended bead slurry to each tube.
- Incubate the tubes at 4°C for 1 hour with gentle agitation.
- Pellet the beads by centrifugation for 10 seconds at 14,000 x g.
- Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.
- Wash the bead 3 times with 0.5 mL of 1X lysis buffer, centrifuging and aspirating each time.
- After the last wash, pellet the beads and carefully remove all the supernatant.
- Resuspend the bead pellet in 40 µL of 2X reducing SDS-PAGE sample buffer.
- Boil each sample for 5 minutes.
- Centrifuge each sample for 10 seconds at 14,000 x g.
Q: How can I get the best results with these beads?
A: To get the best results with these beads, it is important to first determine the amount of cell lysate that is detectable on the blot before performing the pull down. We recommend running a lysate titration on a western blot to determine the concentration that gives a good signal. For the GTPase assay, you will then want to add 100-fold that amount. For example, if you run 5, 10 and 20ug of lysate on a Western blot and 10ug gives a good signal, you will use 10ug x 100 = 1mg of lysate per pull down.
Q: I used the recommended amount of lysate. Why don't I see the active form in the pulldown?
A: The activity level of the small GTPase in the sample will determine how much gets pulled down. The beads are designed to only pull down small GTPase in the GTP form. If the majority of the GTPase in the sample is in the GDP form (inactive), it will not get pulled down, regardless of the amount of lysate loaded. The lysate can be preloaded with GTPgS and used as a positive control.