PAK Beads, Rac1/Cdc42 binding domain protein on beads

Question 1: How much of the beads should I use for my pull-down experiments?

Answer 1: PAK-PBD-GST beads ((Cat. #PAK02) will bind to Cdc42 or Rac1-GDP with a much lower affinity than Cdc42 or Rac1-GTP. If too many PAK-PBD beads are added to the pull-down assay, there will be significant binding to inactive (GDP-bound) Cdc42 or Rac1. The result of this will be an underestimation of Cdc42 or Rac1 activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Cdc42 or Rac1 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 2: How can I test whether the beads are working properly?

Answer 2: A standard biological assay for PAK-PBD GST protein beads consists of a Rac or Cdc42 protein pull-down from cells loaded with either GTP&gamma, S (Cat. #BS01) or GDP. Here are guidelines to follow (see PAK02 datasheet 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 Rac1 or 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 Rac1 or 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 Rac1 or Cdc42 with GDP will inactivate Rac1 or Cdc42 and this complex will bind very poorly to PAK-PBD beads.