Cytoskeleton Signal-Seeker™: Tech Tips and FAQs

Cytoskeleton Signal-Seeker™: Tech Tips and FAQs Tip 1: Pre-Clearing lysate with control beads Tip 2: Optimizing antibody conditions for western immunoblotting Tip 3: Choosing the right lysis buffer for PTM IP Tip 4: Heavy and light chain background interference from Host IgG Tip 5: Rare occurances of de-PTM inhibitors non-specifically modifying target proteins Tip 6: High background and "ghost bands" from primary PTM antibodies for western immunoblotting when using fluorescent secondary antibodies Question 1: Why should I use Signal-Seeker™ PTM detection kits rather than a protein specific antibody for PTM IP detection?
Pre-clearing lysate with control beads Tip #1: Some proteins have non-specific interactions with the Fc region of antibodies as well as the affinity matrix to which the beads are attached. In this case a pre-clearing step may aide in decreasing the non-specific signal from the IP samples. Tip #2: Signal-Seeker control beads are an ideal tool for pre-clearing lysate, because they were developed with null binding-domains/antibodies covalently linked to the appropriate affinity matrix to best recapitulate non-specific interactions. Tip #3: Using naked control beads may not capture all non-specific interactions, because the non-specific binding may occur to binding domains/antibodies, and supplementing with binding-domains/IgG may result in uninterpretable results if they are not effectively removed prior to IP with target antibody-affinity matrix.
Optimizing antibody conditions for western immunoblotting Tip #1: Optimization of primary and secondary antibody for western immunoblotting of IP samples may be necessary because PTM-modified-IP signal for a target protein may be significantly less than total protein signal. Tip #2: Consider optimizing, buffer, blocking reagent, concentration of primary and/or secondary antibody, as well as incubation time for primary and/or seondary antibody.
Choosing the right lysis buffer for PTM IP Tip #1: Certain protein modifications such as SUMOylation require strong denaturing buffer lysis to preserve the PTM modification profile (Figure A); conversely, other protein modifications such as ubiquitination isolation are performed using affinity reagents that are sensitive to denaturing lysates (Figure B). This makes studying crosstalk between these modfications difficult using the same lysate. Tip #2: Cytoskeleton Inc. developed a "universal" buffer system that maintains a robust signal for SUMOylated modified proteins (Figure C), while still effectively capturing a robust ubiqutination signal (Figure D). This will save time and resources by allowing users to study multiple PTMs using the same lysis system.
Heavy and light chain background interference from Host IgG Tip #1: When working with tissue lysate, proper perfusion of the tissue is essential to minimize host-IgG contamination. Tip #2: The figure to the right shows an example where RhoGDI acetylation is being investigated in mouse tissue. Utilizing a mouse primary antibody followed by mouse-HRP secondary results in significant heavy and light chain background signal (panel A). The signal is due to host-IgG contamination as the signal appears in the input, and is visualized with mouse-HRP alone (panel D). Tip #3: Possible approaches to minimize visualization of Host-IgG background signal is to utilize a primary antibody from a different host (panel B), use a specialized secondary antibody like cleanblot (panel C) that recognizes intact IgG, or trying pre-clearing approaches (panel A). We recommend, when possible, to utilize antibodies that are different from the host tissue. Clean blot significantly decreases IgG visualization, but also reduces signal (see input panel C). Pre-clearing is not sufficient at removing all host-IgG contamination.
Rare occurances of de-PTM inhibitors non-specifically modifying target proteins Tip #1: Signal-Seeker tools have made a meaningful step forward in PTM investigation by enabling investigators to study multiple PTMs using a single lysis system and a validated combination of de-PTM inhibitors. Tip #2: Under rare circumstances it is possible to observe non-specific reactivty of the de-PTM inhibitors. The figure to the right shows an example where PDHA1 appears to be artificially modified in the presence of the cocktail of de-PTM inhibitors as the protein appears to be shifted (Figure A, compare lanes 1 vs 3). Additionally, this artifical modification inhibits detection of acetylated PDHA1 using acetyl-lysine affinity beads (Figure B, compare lanes 1 vs 3). Tip #3: While these off-target effects of the de-PTM inhibitors are rare, it is recommended to confirm individual PTM results in the presence of essential de-PTM inhibitors necessary to preserve the PTM being investigated. For example, when investigating PDHA1 acetylation we recommend performing the experiment with lysate plus PIC02 and HDAC inhibitors (TSA and Nic) alone to validate results.
PTM antibodies may result in high background with fluorescent secondary antibodies Tip #1: Signal-Seeker PTM antibodies may produce high background and/or "ghost bands when used with fluorescent secondary antibodies. Tip #2: Blocking buffer optimization can significantly reduce high background and the appearance of ghost bands. Specifically, Intercept Blocking Buffer has been shown to reduce high background. Importantly, some blocking solutions (e.g. casein) may not be suitable as they may interfere with PTM detection (e.g. phosphorylation). Tip #3: Optimization of primary and secondary antibody concentrations may significantly reduce high background. A 5-10x reduction in primary concentration may be necessary to reduce background levels, while having minimal impact on PTM detection when using fluorescent secondaries.
Question 1:Why should I use Signal-Seeker™ PTM detection kits rather than a protein specific antibody for PTM IP detection? Answer 1:Protein specific antibody IP for PTM identification should be used for validation rather than for initial discovery. There can be several pitfalls when using protein specific antibody IP, such as, whether the antibody is IP compatible, will the PTM modification interfere with antibody binding, is the antibody compatible with a particular lysis buffer, as well as, whether or not to conjugate to beads in order to minimize heavy and light chain contamination. Signal-Seeker™ PTM detection kits have been developed to minimize the need to address these optimization questions in order for the user to focus on obtaining critical, and meaningful results.

Fuente:Cytoskeleton