Cytoskeleton/RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays/3 x 10 Assays/BK030

Question 1: I have high background and/or multiple bands on my western blot. How can I fix this?

Answer 1: There are multiple causes of high background and/or multiple bands. Some suggestions to improve background signal include:

1. When blotting use 70v for 45min only as the small G-proteins are very mobile.
2. Fully remove SDS from the gel by using a non-SDS containing buffer for transfer and performing a full 15 min gel wash step in the transfer buffer before blotting.
3. Dry the PVDF membrane for 30 min after transfer and before blocking (not necessary for nitrocellulose)
4. Making sure that the TBST contains 10 mM Tris, 0.05% Tween 20 and 150 mM NaCl.
5. Incubating with the primary antibody overnight at 4°C and using the appropriate ECL detection system.

Question 2: How much of the beads should I use for my pull-down experiments?

Answer 2: The beads conjugated to the respective effector protein that recognizes the active form of each GTPase will bind to the GDP-bound GTPase with a much lower affinity than the GTP-bound GTPase. If too many beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) GTPases. The result of this will be an underestimation of GTPase activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given GTPase activation or inactivation assay. Once optimal conditions have been established, bead titrations should no longer be necessary.

Question 3: How can I test whether the beads are working properly?

Answer 3: A standard biological assay for the beads consists of a GTPase protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP. Here are guidelines to follow (see Cat. # BK030 manual for more details):

Positive Cellular Protein Control:

Total cell lysate (300 – 800 μg) should be loaded with GTPγS as a positive control for the pull-down assay. The following reaction details how to load endogenous GTPase with the nonhydrolysable GTP analog (GTPγS). This is an excellent substrate for the beads and should result in a strong positive signal in a pull-down assay.

a) Perform GTP loading on 300 – 800 μg of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer.

b) Immediately add 1/100th volume of GTPγS (200 μM final concentration). Under these conditions, 5 - 10% of the GTPase protein will load with non-hydrolysable GTPγS and will be “pulled-down” with the beads in the assay.

c) Incubate the control sample at 30°C for 15 min with gentle rotation.

d) Stop the reaction by transferring the tube to 4°C and adding 1/10th volume of STOP Buffer.

e) Use this sample immediately in a pull-down assay.

Negative Cellular Protein Control:

This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTPγS. Loading endogenous GTPase with GDP will inactivate the GTPase and this complex will bind very poorly to the beads.

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com