THE NEWSLETTER OF ACTIVE MOTIF — November 2006 • volume 7 • number 4 New: Magnetic Beads Make Chromatin INSIDE THIS ISSUE Immunoprecipitation Faster and Easier 2 ELISA for Quantifi cation of Activated Ras GTPase Active Motif’s new ChIP-IT™ Express Kits use protein G-coated magnetic beads Simplify Co-Immunoprecipitation instead of traditional agarose beads to make chromatin immunoprecipitation 3 (ChIP) simpler and faster than ever before. These innovative beads have made 4 Investigate SUMOylation with Simple, Effi cient SUMOlink™ it possible to simplify and streamline the protocol, optimize buffers and 5 New: Study Your Choice of reduce or eliminate several time- and labor-intensive steps. The result is that Target in Any Cell Signaling ChIP-IT Express enables you to perform ChIP in a single day. Pathway 7 TransAM™: The Simple Way to Assay Transcription Factor New technique for speed and success ously elute your DNA from the beads Activation The new components and protocol while reversing the cross-links. These of ChIP-IT Express make it faster and simple protocol changes give you results 8 Simplifi ed Analysis of easier than any other ChIP procedure. quickly and easily while offering the DNA Methylation The kits include all buffers, reagents freedom to simultaneously perform and components required to shear 5 multiple ChIP assays: now you can even samples of chromatin, using your choice perform ChIP in PCR tubes with a multi- of sonication or enzymatic digestion, channel pipettor (Figure 1). then perform 25 ChIP reactions. We’ve even included a strong bar magnet. The The power of magnetic beads protein G-coated magnetic beads, pro- With ChIP-IT Express Kits, ChIP can vided ready-to-use, have a high binding be performed in the included Eppen- capacity for IgG. This enables them to dorf tubes or in 8-well PCR strips. PCR be used in small volumes, which reduces strips make it convenient to process non-specifi c binding. This improved larger numbers of samples. The protein specifi city enables the number and G-coated magnetic beads pellet in length of washing steps to be reduced. It seconds, much faster than centrifuging is not even necessary to block the beads agarose beads. An added advantage is or pre-clear the chromatin. that magnetic beads pellet on the tube side instead of the bottom, even coming Optimized buffers eliminate steps completely out of the wash buffer. This Figure 1: Multiple-sample ChIP using ChIP-IT Express. Washing magnetic beads is fast and easy because the In addition to the magnetic beads and makes it easy to remove buffers without pellet forms against the side of the tube in seconds. bar magnet, ChIP-IT Express Kits contain disturbing the beads, which reduces This makes it possible to ChIP multiple samples in new buffers that eliminate the need for sample loss and helps ensure sample-to- 8-well PCR tubes using a multi-channel pipettor. DNA purifi cation, so you can simultane- sample consistency. continued on page 6 www.activemotif.com 1 Small GTPase Activation ELISA for Quantifi cation of Activated Ras GTPase Active Motif’s Ras GTPase Chemi ELISA Kit is the fi rst ELISA-based kit Ras ELISA advantages designed to detect and quantify activated Ras GTPase. The method offers • More sensitive – assay uses only 25 µg of extract, or 20-fold less several advantages over other commercially available kits, which require you than pull-down/Western methods to perform immunoprecipitation of Ras, followed by Western blotting. • Better results – quantitative data makes it easier to compare results In contrast, the Ras GTPase Chemi ELISA as a probe to isolate activated Ras. The • Less effort – no need to perform IP, uses Raf-RBD protein and antibodies in Ras ELISA Kit contains a Raf-RBD protein run gels or develop Western blots a 96-well format to capture and quan- fused to GST and a 96-well, glutathione- • Save time – results in < 5 hours tify the activated Ras in your sample. coated assay plate. GST-Raf-RBD is fi rst • Versatile – assay activated extracts This faster, more sensitive alternative incubated on the plate for one hour to from cells or tissue samples, or enables you to use less of your precious immobilize this capture protein. Addi- study recombinant Ras protein sample, yet still detect low-level events. tion of sample to the plate results in the In addition, because ELISAs provide more binding of activated Ras to the Raf-RBD. 800000 Unstimulated quantitative results than Westerns, the A primary antibody specifi c for Ras is EGF stimulated data generated is more meaningful. then added, followed by an HRP-conju- 600000 gated secondary antibody and develop- 400000 ed Ras Signal (RLU) The Ras GTPase Chemi ELISA method ing reagent (Figure 1). The plate is then at Activ 200000 Because activated Ras binds specifi cally read on a luminometer, which provides a 0 to the Ras-binding domain (RBD) of the sensitive, quantitative chemiluminescent 6.25 12.5 25 50 Raf effector protein, Raf-RBD is used readout of activated Ras (Figure 2). Cell extract (µg/well) Figure 2: Quantifi cation of activated Ras. Increasing amounts of whole-cell extract from HeLa cells that had been stimulated with 5 ng/ml EGF for 2 minutes “ELISAs are faster and more sensitive than other methods used to were assayed using the Ras GTPase Chemi ELISA Kit. study GTPase activation, and eliminate the need for IP, gels and blots.” Try the quantitative, sensitive assay The Ras GTPase Chemi ELISA Kit makes Add primary Add anti-IgG antibody HRP conjugate it fast and easy to detect and quantify 45 min activated Ras GTPase. The kit is ideal 45 min 45 min for the study of novel signaling path- Add cell Add developing extract to solution, then ways that activate Ras, as well as for plate quantify determining if a particular malignancy is related to inappropriately activated, oncogenic Ras. Please give us a call, re- turn the enclosed reply card or visit our website at www.activemotif.com/gtpase Cell extract containing activated GST-Raf-RBD for complete information, including Ras GTPase coated plate downloadable manuals. To get to the Figure 1: Flowchart of the Ras GTPase Chemi Kit. best Ras activation assay available, try Cell extract is added to a glutathione-coated plate that contains immobilized GST-Raf-RBD protein. Activated Ras in the the Ras GTPase Chemi ELISA Kit. extract binds to the Raf-RBD protein. Addition of primary & secondary antibodies and developing solution followed by reading on a luminometer enables sensitive quantification of activated Ras. Product Format Catalog No. Ras GTPase Chemi ELISA Kit 1 x 96-well plate 52097 2 Toll Free — 1 877 222 9543 November 2006 • volume 7 • number 4 Co-Immunoprecipitation Simplify Co-Immunoprecipitation Active Motif’s Nuclear Complex Co-IP Kit simplifi es co-immunoprecipitation studies of nuclear protein complexes by providing you with optimized reagents 250 for both nuclear extract preparation and immunoprecipitation. 1 2 3 The Nuclear Complex Co-IP’s extraction bound, interacting proteins. The complex Figure 1: Western blot analysis of the IP’d p33 subunit of the RNA pol II complex. process provides a simple and effective is then detected by Western blot using a HeLa cells were grown to confluence on 100 mm plates method to obtain and maintain protein second antibody targeted against one of and nuclear extracts were prepared using the kit’s complexes contained in nuclear compart- the bound, interacting proteins. However, extraction reagents. For IP experiments, the stringency of the IP High Buffer was increased by supplementing ments of the cell, specifi cally those previ- traditional methods for performing Co-IP with NaCl and Detergent. 100 µg of nuclear extract was ously bound to DNA, while the versatile are not optimal for studying complexes of used per IP reaction and incubated with either 2 µg p33 antibody or no antibody. Following the IP, Western Co-IP reagents offers you the fl exibility DNA-binding proteins as these complexes, blot analysis was performed using RNA pol II mouse to vary the stringency of the Co-IP buffer being fragile, are frequently disrupted mAb at 0.1 µg/ml followed by anti-mouse HRP at 1:1000. compositions. This improves your results during the extraction process. In addition, Detection of the p33/RNA pol II complex by the RNA pol II antibody (lane 3) demonstrates that the Co-IP and enables you to study tightly bound many protein complexes are altered by was successful in maintaining the protein complex. The or weak protein complexes with ease. the salt and detergent composition of the input HeLa extract (lane 1) was run as a control for the buffers used in the immunoprecipitation Western blot using 0.1 µg/ml RNA pol II. Co-Immunoprecipitation (Co-IP) is often process, which can complicate their Lane 1 Western blot control Lane 2 Negative Control (no antibody used in IP) used to fi nd and study protein/protein analysis. To overcome these problems, Lane 3 Co-IP: IP using p33/WB using RNA pol II interactions. In Co-IP, a fi rst antibody the Nuclear Complex Co-IP Kit extraction is used to immunoprecipitate a target reagents were designed to help maintain The Co-IP Kit also contains high and low antigen, which also co-precipitates any nuclear protein complexes. stringency IP buffers, as well as salt and detergent. Addition of salt and detergent is ideal for robust protein/protein inter- “The Nuclear Complex Co-IP Kit simplifi es co-immunoprecipitation of DNA- actions because it reduces background. binding proteins from cell and tissue samples by providing both extraction and However, as unstable protein complexes immunoprecipitation components that maintain nuclear protein complexes.” may not withstand high stringencies, this convenient kit format enables stringency to be modifi ed as required for each Cell nucleus containing the two Nuclear extraction Add antibody directed against Add antibody particular protein complex.