The Ras family of small GTPases consists of at least 35 related human proteins that include the oncoproteins HRAS, KRAS and NRAS as the founding members (Colicelli 2004). The Ral proteins, RalA and RalB, share 58% identity to these oncoproteins and 85% identity with each other (Chardin and Tavitian 1986). Ral proteins play an important role in diverse cellular processes including endocytosis, exocytosis, oncogenesis and the regulation of transcription and cell morphology (Feig 2003)Like other small GTPases, Ral proteins become activated when they switch from the GDP-bound state to the GTP-bound state (Takai, Sasaki and Matozaki 2001), and it is the GTP-bound form that specifically interacts with their downstream effector proteins. The fact that Ral family effector proteins will specifically recognize the GTP-bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors Ral protein activation (Hofer, Berdeaux and Martin 1998). The assay uses the Ral Binding Domain (RBD) of the effector protein RalBP1. The RBD protein motif has been shown to bind specifically to the GTP-bound form of Ral. The fact that the RBD region of RalBP1 has a high affinity for GTP-Ral makes it an ideal tool for affinity purification of GTP-Ral from cell lysates. The RalBP1-RBD protein supplied in this kit has been expressed as an His-tagged fusion protein in E. coli. and bound to colored beads. This allows one to “pulldown” GTP-Ral complexed with Ral-BP1-RBD beads. This assay provides a simple means of analyzing cellular RalA activities in a variety of systems. The amount of activated RalA is determined by a Western blot using a RalA specific antibody.
The kit contains sufficient materials for 50 assays and includes reagents for positive and negative controls. The following components are included:
Chardin, P., and A. Tavitian. "The ral gene: a new ras-related gene isolated by the use of a synthetic probe." EMBO J 5 (1986): 2203-2208.
Colicelli, J. "Human RAS Superfamily Proteins and Related GTPases." Sci STKE 250, no. Re13 (2004): 1-53.
Feig, L.A. "Ral-GTPases: approaching their 15 minutes of fame." Trends in Cell Biology 13, no. 8 (2003): 419-425.
Hofer, F., R. Berdeaux, and G.S. Martin. "Ras-independent activation of Ral by a Ca2+-dependent pathway." Current Biology 8 (1998): 839-842.
Takai, Y., T. Sasaki, and T. Matozaki. "Small GTP-binding proteins." Physiol. Rev. 81 (2001): 153-208.
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Figure 1. RalBP1-RBD bead pulldown Assays. Rat2 cells were grown to 70% confluency in DMEM media supplemented with 10% fetal calf serum (FCS) prior to 48h growth in serum free media. Cell lysates were processed as described in Section VI of this manual. Active RalA was purified from 400 µg of cell lysates by incubating lysates with 10 µg of RalBP1-RBD beads as described in section VI. All bead samples were resuspended in 10 ul of 2x sample buffer and run on a 4-20% SDS gel. Protein was transferred to PVDF, probed with a 1:1000 dilution of anti-RalA and processed for chemiluminescent detection of RalA as described in Section VI: STEP 4. Lane 1: 20 ng recombinant RalA-His control protein; Lane 2: bead bound RalA from lysates of cells treated for 2 minutes with Epidermal growth factor (100mg/ml); Lane 3: bead bound RalA from lysates of cells grown in serum free media for 48h. Densitometric analysis of chemiluminescent signal showed that RalA activation in EGF treated cells was 2 fold above RalA activation in serum starved cells.
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:
Question 2: How much of the beads should I use for my pull-down experiments?
Answer 2: RalBP1-RBD beads (Part # RL07) will bind to RalA-GDP with a much lower affinity than RalA-GTP. If too many RalBP1-RBD beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) RalA. The result of this will be an underestimation of RalA activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given RalA activation or inactivation assay. Once optimal conditions have been established, bead titrations should no longer be necessary. We recommend 5, 10 and 20 μg bead titrations.
Question 3: How can I test whether the beads are working properly?
Answer 3: A standard biological assay for RalBP1-RBD beads consists of a RalA protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP. Here are guidelines to follow (see Cat. # BK040 datasheet for more details):
Positive Cellular Protein Control:
Total cell lysate (200 – 400 µg) should be loaded with GTPgS as a positive control for the pull-down assay. The following reaction details how to load endogenous RalA with the nonhydrolysable GTP analog (GTPgS). This is an excellent substrate for RalBP1-RBD beads and should result in a strong positive signal in a pull-down assay.
a) Perform GTP loading on 200 – 400 μg of cell lysate by adding 1/10th volume of Loading Buffer.
b) Immediately add 1/100th volume of GTPgS (200 μM final concentration). Under these conditions, 5 - 10% of the RalA protein will load with non-hydrolysable GTPgS and will be “pulled-down” with the RalBP1-RBD 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 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 GTPgS. Loading endogenous RalA with GDP will inactivate RalA and this will bind very poorly to RalBP1-RBD beads.
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