The Rac1 Activation Assay Kit from Upstate is designed to identify the amount of active Rac (i.e. Rac-GTP, rather than the inactive Rac-GDP) within a cellular lysate, from either adherent or non-adherent cells. It utilizes a glutathione (GST) pull-down method, with GST-agarose beads bound to a portion of p21-activated kinase (PAK-1) containing amino acids 74-89 of the N-terminal regulatory region. This region of PAK-1, known as the p21 binding domain (PBD), interacts specifically with the active form of Rac1, thus providing an excellent tool for isolating active Rac1 in cellular samples. Cells are lysed using magnesium lysis buffer (provided); the lysates are then pre-cleared with GST-agarose beads (not provided). At this step, a small amount of lysate can be saved to measure the amount of protein; positive and negative controls can also be prepared using the GTP and GTP-gamma-S reagents provided with the kit. Next, the PAK-1 PBD beads are added to the cell lysates (10 ul of beads for every 500 ul of cell lysate). Following incubation to allow binding of Rac1-GTP, the beads are washed with buffer to remove cellular debris and unbound Rac1. Finally, laemmli buffer (not provided) is added to the beads and the slurry is run in a Western blot using the provided anti-Rac1 antibody.
In my research, I have used this kit on adherent cells (human aortic endothelial cells and mouse aortic smooth muscle cells) and non-adherent cells (mouse lymphocytes), as well as whole tissue samples (pig heart tissue). The provided protocols for adherent and non-adherent cells are very clear; however, there are no instructions for analyzing Rac1-GTP in tissues. For this, I had to optimize my own method which involved testing different amounts of tissue. In my experiments, I used 30 mg of frozen, crushed tissue powder mixed with 550 ul of the lysis buffer, followed by homogenization in a cold glass Dounce homogenizer. These methods were the most suitable for detecting Rac1-GTP with tissues (Dudley et al, Circulation, 112(9):1266-73, 2005.
Throughout my numerous (nearly 50) uses of this kit, I identified some protocol changes which can improve the outcome. The biggest change that I recommend is that all steps (yes, ALL steps) of the pull-down be performed in a cold (4°C) room. Although uncomfortable, this greatly reduces the risk of Rac-GTP hydrolysis to Rac-GDP and thus reduces variability when repeating experiments. It is also important to thoroughly mix the bead and laemmli slurry before loading to the gel. This will reduce the chance of unequal loading of samples. Since the protocol recommends adding 500 ul of each lysate for the pull-down assay, I suggest using no more than 600 ul of lysis buffer for cells or tissue (75-80% confluent 100 mm dish of adherent cells, 107 non-adherent cells, or 30-50 mg tissue powder). This is optimal because it concentrates the lysate while making sure the lysate volume is sufficient for the pull-down assay. After the very last wash, it is extremely difficult to remove all of the buffer without removing beads as well. So, I recommend using a 28-gauge needle syringe (insulin syringe) to fully remove all of the buffer from the beads. Any needle larger than this will take up beads, so make sure it is an insulin syringe. And finally, when preparing to incubate the membrane with the primary antibody, it is best to cut the membrane near 30 kD and incubate only the bottom part of the membrane. This removes the 50 kD “additional unknown protein” that the antibody detects, which can shine very brightly and bleed into the Rac1-GTP band.
Overall, the kit works well, especially the primary antibody, and provides reliable results. Before using this kit, I had tried assessing Rac1 activity by making membrane and cytosolic fractions from cell lysates and using Western blotting methods. Using that method was never truly consistent, so I switched to using Upstate’s kit and am quite happy with the change.
Nyssa E. Hoch
Graduate Student
Emory University
Division of Cardiology