Pull-down Assays

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com.

Many publications cite the use of Cytoskeleton's kits in the Materials and Methods section of papers. Usually the citation is associated with a particular result in the form of a graph or image that helps the you, the authors, present your findings. This indicates the utility of the Kits to produce publication quality data in a short timeframe thus helping improve the productivity of your efforts. Example citations for pull-down assay kits are shown below.  More citations are available on individual product pages.



Question 1:  Why does Cytoskeleton still provide pull-down assays when G-LISA technology is clearly superior?

Answer 1:  At Cytoskeleton, we strive to be the one-stop provider for small G-protein activation assays, whether they are the traditional pull-down or the new G-LISA assays.  The pull-down assays are useful when a researcher wants to do a “quick look and see” experiment involving a limited number of conditions (1 time point and 1 drug concentration or treatment condition) with a limited number of samples.  Also, some researchers are more comfortable with the traditional pull-downs or need to do some new pull-down assays to directly compare with previous pull-down data. 

Question 2: Why do I get doublet bands in some experiments and not in others?

Answer 2:  Sometimes researchers have reported that when using our Cdc42 or Rac1 pull-down activation assays (Cat. # BK034 and BK035, respectively), the GTPase signal appears as a doublet band. The most sensitive point for doublet formation is after resuspending the beads in sample buffer, and is caused by insufficient denaturation of proteins prior to SDS-PAGE chromatography. The solution is to use double the amount of 2 x sample buffer i.e. 40 µl, to resuspend the pellet, and to boil them for 1min. Samples can be stored at -20°C and analysed at a later date.  1 x Sample buffer is 63 mM Tris pH 6.8, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue.

For more information, check out the manuals in the Document tab, or e-mail tservice@cytoskeleton.com for more in-depth questions.