TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>Immunoprecipitation (IP) is a valuable tool that utilizes antibody: antigen interactions to isolate a specific target protein from thousands of proteins in a complex solution. The specificity of the reaction increases the concentration of dilute proteins as high as 10,000-fold; a property that renders the technique useful in the examination of rare proteins. With careful choice of antibodies, a co-IP can also be used to demonstrate protein interaction partners.</p>
</div>
</div>
</div>

[GUIDELINES]
<p><strong>I. Introduction</strong></p>
<p> </p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p>Immunoprecipitation (IP) is a valuable tool that utilizes antibody: antigen interactions to isolate a specific target protein from thousands of proteins in a complex solution. The specificity of the reaction increases the concentration of dilute proteins as high as 10,000-fold; a property that renders the technique useful in the examination of rare proteins. With careful choice of antibodies, a co-IP can also be used to demonstrate protein interaction partners*.</p>
<p> </p>
<p>The typical workflow for an IP can be divided into four parts:</p>
<ol>
<li>
<p>Incubation of the primary antibody with the sample (e.g., a cell lysate);</p>
</li>
<li>
<p>Capture of the antibody: antigen complex using a solid support such as Protein A agarose beads;</p>
</li>
<li>
<p>Separation of the proteins by SDS-PAGE;</p>
</li>
<li>
<p>Detection of the target protein by Western blot.</p>
</li>
</ol>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p> </p>
<p>The technique is time-consuming and often requires optimization of antibody and lysate concentrations as well as the inclusion of appropriate positive and negative controls. Ultimately, success relies on the choice of antibodies, not only for the IP step but also for Western blot detection of post-IP proteins. A common solution to avoid detection of the heavy and light chains from the IP step in the Western blot is to use two separate antibodies to the same target but from different host species. An example would be using a mouse capture antibody for IP and a rabbit antibody for post-IP Western blot detection. The LI-COR Quick Western Kit-IRDye 680RD eliminates the need for two separate antibodies in the IP workflow. The proprietary Detection Reagent does not recognize denatured heavy or light chains from monoclonal antibodies; therefore, the same monoclonal antibody can be used for the IP step AND post-IP Western blot detection. In addition, the Quick Western Protocol saves 90 minutes of assay time compared to a traditional two-step Western blot protocol*.</p>
<p> </p>
<div class="page" title="Page 2">
<div class="layoutArea">
<div class="column">
<p><em>*NOTE: The data, procedure, and troubleshooting outlined in this Technical Note are provided as guidelines. A complete discussion of the IP technique and the Western blot technique are beyond the scope of this material.</em></p>
<p> </p>
<div class="page" title="Page 3">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The following data simulate post-IP Western blot detection utilizing traditional IRDye secondary antibody detection (panel A) compared with Quick Western Kit-IRDye 680RD detection (panel B). The traditional detection method recognizes the denatured heavy and/or light chains of the IP antibodies. The Quick Western Kit detection method does not recognize any denatured heavy and light chains from the IP antibodies evaluated. This lack of recognition is the foundation for single antibody IP and post-IP Western blot detection.</p>
<p> </p>
<div class="page" title="Page 3">
<div class="section">
<div class="layoutArea">
<div class="column">
<p><em>NOTE: The Detection Reagent will recognize denatured heavy or light chains of certain polyclonal antibodies. It will not recognize denatured heavy or light chains of monoclonal (mouse or rabbit) antibodies.</em></p>
<p> </p>
<p><em><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hf9c2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hf9c2w6.png" data-ofn="Screen Shot 2016-12-29 at 7.19.51 AM.png" /></em></p>
<p> </p>
<p><strong>II. Required Reagents</strong></p>
<div class="page" title="Page 3">
<div class="section">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>Quick Western Kit-IRDye 680RD (LI-COR, P/N 926-68100)</p>
</li>
<li>
<p>A431 Cell Lysate in 50 mM Tris-HCl pH7.8, 150 mM NaCl, 1% NP-40, 0.2% Sodium Deoxycholate, 1X Halt Protease Inhibitor</p>
</li>
<li>
<p>Protein A Beads (Thermo Fisher, P/N 20333)</p>
</li>
<li>
<p>Anti-ERK2 Mouse Antibody (Santa Cruz Biotechnology, P/N SC1647)</p>
</li>
<li>
<p>1XPBS</p>
</li>
<li>
<p>Tween® 20</p>
</li>
<li>
<p>4X Protein Sample Loading Buffer (LI-COR, P/N 928-40004). Dilute to 2X with water.</p>
</li>
<li>
<p>26.5-gauge needle</p>
</li>
<li>
<p>3 mL syringe</p>
</li>
<li>
<p>20X MES Buffer (Invitrogen, P/N NP0002)</p>
</li>
<li>
<p>NuPAGE® Antioxidant (Invitrogen P/N NP0005)</p>
</li>
<li>
<p>NuPAGE 10% Bis-Tris gels (Invitrogen P/N NP0303BOX)</p>
</li>
<li>
<p>Odyssey® Protein Molecular Weight Marker (LI-COR® P/N 928-40000)</p>
</li>
<li>
<p>Tris-Glycine Buffer</p>
</li>
<li>
<p>Nitrocellulose (LI-COR P/N 926-31092)</p>
</li>
<li>
<p>Odyssey Blocking Buffer (PBS) (LI-COR P/N 927-40000)</p>
</li>
<li>
<p>IRDye 680RD Goat anti-Mouse (LI-COR P/N 926-68070)</p>
</li>
</ul>
<p> </p>
<p><strong>III. Sample Protocol: Immunoprecipitation and Detection of ERK2</strong></p>
<p> </p>
<div class="page" title="Page 4">
<div class="layoutArea">
<div class="column">
<p><em>NOTE: This is intended as a general guideline for an IP and Western blot procedure. Different targets will require optimization of antibody concentrations, lysate concentrations, and incubation times. In addition, solid supports other than Protein A agarose beads may be substituted.</em></p>
<p> </p>
<p><em>IMPORTANT: It is critical that the antibody chosen is recommended for both immunoprecipitation AND Western blot by the primary antibody vendor. This information can be found in the primary antibody pack insert or on the vendor’s website.</em></p>
<p> </p>
<p><em>WARNING: Mouse IgG1 subclass antibodies may not work in this procedure, or may require additional optimization.</em></p>
</div>
</div>
</div>
<p> </p>
<p>See 'STEPS' for protocol. </p>
</div>
</div>
</div>
<div class="section"> </div>
<div class="section"><strong>IV. Experimental Results</strong></div>
<div class="section"> </div>
<div class="section"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hgac2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hgac2w6.png" data-ofn="Screen Shot 2016-12-29 at 7.24.12 AM.png" /></div>
<div class="section"> </div>
<div class="section"><strong>V. Experimental Considerations</strong></div>
<div class="section">
<div class="page" title="Page 8">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>Negative and positive controls are extremely important for correct interpretation of the data and should ALWAYS be included in an immunoprecipitation experiment.</p>
</li>
<li>
<p>Careful consideration should be given in the choice of solid support. Be sure that your selection provides the best binding affinity to the type of antibody used. Protein A-sepharose, agarose, magnetic beads, etc., are best for mouse IgG2a, IgG2b, or rabbit IgG. Protein G has a higher affinity for mouse IgG1. Consult the manufacturer’s guide if you are uncertain of the best support for</p>
<p>your experiment.</p>
</li>
<li>
<p>Mouse or rabbit monoclonal antibodies are recommended for use with this kit (for IP and detection of post-IP proteins by Western blot). The detection reagent recognizes denatured heavy and/or light chains of certain polyclonal antibodies, which may interfere with post-immunoprecipitation detection by Western blot.</p>
<div class="page" title="Page 8"> </div>
</li>
<li>
<p>If there are cross-reacting bands in the lysate + PBS (negative control) as well as your lysate + antibody lanes, pre-clearing the lysates is an option to reduce non-specific contaminants and remove proteins with high affinity to Protein A or G.</p>
</li>
</ul>
</div>
</div>
</div>
<p>           –  Wash an amount of your solid support equal to 30 μL of 50:50 slurry per sample a minimum of 3 times in 1 mL 1X PBS.</p>
<p> </p>
<p>           –  Resuspend beads in the starting volume and add 30 μL to each tube of lysate (step 1), including no antibody control tubes (step 3), and incubate 1 hour at room temperature.</p>
<p>         </p>
<p>           –  Centrifuge on highest speed for 1 minute in a microcentrifuge. Transfer the supernatant to a clean tube using a 26.5-gauge needle and syringe.</p>
           </div>
<div class="section">           –  Start the protocol at Step 2.</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
SECTION: Sample Preparation (Day 1)
?. 
SECTION: Sample Preparation (Day 1)
?. 
SECTION: Sample Preparation (Day 1)
?. 
SECTION: Sample Preparation (Day 1)
?. 
SECTION: Sample Preparation (Day 1)
?. 
SECTION: Sample Preparation (Day 1)
?. 
SECTION: Bead Preparation (Day 2)
?. 
SECTION: Bead Preparation (Day 2)
?. 
SECTION: Bead Preparation (Day 2)
?. 
SECTION: Bead Preparation (Day 2)
?. 
SECTION: Bead Preparation (Day 2)
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Bead Preparation (Day 2)
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Pull Down of Bead:Protein Complex
?. 
SECTION: Electrophoresis and Transfer
?. 
SECTION: Electrophoresis and Transfer
?. 
SECTION: Electrophoresis and Transfer
?. 
SECTION: Electrophoresis and Transfer
?. 
SECTION: Electrophoresis and Transfer
?. 
SECTION: Western Blot Workflow
?. 
SECTION: Western Blot Workflow
?. 
SECTION: Western Blot Workflow
?. 
SECTION: Western Blot Workflow
?. 
SECTION: Western Blot Workflow
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Standard Western Blot Detection
?. 
SECTION: Quick Western Blot Detection
?. 
SECTION: Quick Western Blot Detection
?. 
SECTION: Quick Western Blot Detection
?. 
SECTION: Quick Western Blot Detection
?. 
SECTION: Quick Western Blot Detection
?. 
SECTION: Imaging
?.