Ras Activation Assay Biochem Kit

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: Raf-RBD beads ((Cat. #RF02) will bind to Ras-GDP with a much lower affinity than Ras-GTP. If too many Raf-RBD beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) Ras. The result of this will be an underestimation of Ras activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Ras activation or inactivation assay. Once optimal conditions have been established, bead titrations should no longer be necessary. We recommend 20, 40 and 60 &mu, l (66, 132 and 198 µ, g) bead titrations.

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

Answer 3: A standard biological assay for Raf-RBD beads consists of a Ras protein pull-down from cells loaded with either GTP&gamma, S (Cat. #BS01) or GDP. Here are guidelines to follow (see (Cat. #RF02 or BK008 datasheets for more details):

Positive Cellular Protein Control:

Total cell lysate (200 &ndash, 500 µ, g) should be loaded with GTP&gamma, S as a positive control for the pull-down assay. The following reaction details how to load endogenous Ras with the nonhydrolysable GTP analog (GTP&gamma, S). This is an excellent substrate for Raf-RBD beads and should result in a strong positive signal in a pull-down assay.

a) Perform GTP loading on 200 &ndash, 500 &mu, g of cell lysate that is at a protein concentration between 0.4 &ndash, 2.0 mg/ml by adding 1/10th volume of Loading Buffer.

b) Immediately add 1/100th volume of GTP&gamma, S (200 &mu, M final concentration). Under these conditions, 5 - 10% of the Ras protein will load with non-hydrolysable GTP&gamma, S and will be &ldquo, pulled-down&rdquo, with the Raf-RBD beads in the assay.

c) Incubate the control sample at 37°C for 30 minutes 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 in a pull-down assay immediately.

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 GTP&gamma, S. Loading endogenous Ras with GDP will inactivate Ras and this will bind very poorly to Raf-RBD beads.