Cdc42 Pulldown Activation Assay Kit

AT

he Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state. Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation. The fact that many Rho 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 Rac and Cdc42 protein activation. The assay uses the Cdc42/Rac Interactive Binding (CRIB) region (also called the p21 Binding Domain, PBD) of the Cdc42 / Rac effector protein, p21 activated kinase 1 (PAK). The CRIB/PBD protein motif has been shown to bind specifically to the GTP-bound form of Rac and/or Cdc42 proteins. The fact that the PBD region of PAK has a high affinity for both GTP-Rac and GTP-Cdc42 and that PAK binding results in a significantly reduced intrinsic and catalytic rate of hydrolysis of both Rac and Cdc42 make it an ideal tool for affinity purification of GTP-Rac and GTP-Cdc42 from cell lysates. The PAK-PBD protein supplied in this kit corresponds to residues 67-150. This includes the highly conserved CRIB region (aa 74-88) plus sequences required for the high affinity interaction with GTP-Rac and GTP-Cdc42. The PAK-PBD is in the form of a GST fusion protein, which allows one to "pull-down" the PAK-PBD/GTP-Cdc42 (or GTP-Rac) complex with glutathione affinity beads. The assay therefore provides a simple means of quantitating Rac/Cdc42 activation in cells. The amount of activated Cdc42 is determined by a Western blot using a Cdc42 specific antibody.

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: PAK-PBD-GST beads ((Cat. #PAK02) will bind to Cdc42-GDP with a much lower affinity than Cdc42-GTP. If too many PAK-PBD beads are added to the pull-down assay, there will be significant binding to inactive (GDP-bound) Cdc42. The result of this will be an underestimation of Cdc42 activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Cdc42 activation or inactivation assay. Once optimal conditions have been established, bead titrations should no longer be necessary. We recommend 10, 15 and 20 &mu, g bead titrations.

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

Answer 3: A standard biological assay for PAK-PBD GST protein beads consists of a Cdc42 protein pull-down from cells loaded with either GTP&gamma, S (Cat. #BS01) or GDP. Here are guidelines to follow (see (Cat. #PAK02 or BK034 datasheets for more details):

Positive Cellular Protein Control:

Total cell lysate (300 &ndash, 800 &mu, 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 Cdc42 with the nonhydrolysable GTP analog (GTP&gamma, S). This is an excellent substrate for PAK-PBD beads and should result in a strong positive signal in a pull-down assay.

a) Perform GTP loading on 300 &ndash, 800 &mu, g of cell lysate (0.5 mg/ml protein concentration) 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 Cdc42 protein will load with non-hydrolysable GTP&gamma, S and will be &ldquo, pulled-down&rdquo, with the PAK-PBD 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 GTP&gamma, S. Loading endogenous Cdc42 with GDP will inactivate Cdc42 and this complex will bind very poorly to PAK-PBD beads.

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SDS-PAGE minigel system and western blotting transfer apparatus

The brightly colored glutathione agarose beads in BK034 makes the kit easy to use.