Discovery Through Validation: ChIP Assays


Posted by Carolyn P on Sep 30, 2015 9:43:00 AM

Cell Signaling Technology is proud to present the following On-Demand Webinar: 

The Use of Highly Validated Antibodies and Optimized ChIP Assays to Analyze Epigenetic Marks and Mechanism in DIsease

Sayura Aoyagi, Ph.D, CST Antibody Validation Scientist

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Topics: Cancer Research

History of the Monoclonal Antibody - Part I


Posted by Carolyn P on Aug 26, 2015 8:30:00 AM

 

Monoclonal antibodies are a pretty ubiquitous research tool. In fact, you’d be hard-pressed to find a lab without a freezer full of them. They have myriad uses from examining protein localization within a tissue (e.g., IHC), to isolating a particular protein out of a proteinaceous soup (e.g. WB, IP), or separating a specific cell population from the tissue milieu (e.g., FACS).  They are such a fundamental part of so many research strategies that without them there wouldn’t be many tricks left up our collective sleeve.  So, it’s a little strange to think that we really haven’t had them at our disposal for all that long . . . 

. . . setting the wayback machine for Cambridge, UK  1975 . . . 

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Topics: Just for fun

Need Better ChIP results? Use Enzymatic Digestion


Posted by Carolyn P on Aug 5, 2015 12:33:00 PM

The success or failure of a chromatin immunprecipitation (ChIP) experiment is highly dependent on the integrity of both the chromatin and the protein epitope, so your method of chromatin preparation can have a significant impact on the quality of your results.

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Topics: Protocols, ChIP, techniques

Why High Concentration doesn't always mean good Antibody Performance.


Posted by Carolyn P on Jul 29, 2015 5:00:00 AM

If you buy primary antibodies from different places, you may have noticed that many companies list the size of their products in micrograms per ml (i.e., by weight). You may have also noticed that CST does not - CST antibodies are listed by performance (e.g., 10 western blots) instead.

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Topics: Antibody Performance, Primary Antibodies, techniques

4 Steps to Better Immunohistochemistry: Steps 2, 3, and 4.


Posted by Carolyn P on Jul 15, 2015 7:00:00 AM

This is part two of a two-part series on how to optimize your IHC protocols. Part one introduced the principles behind antigen retrival. Click here if you missed it...but, if you've got your tissue prepped and ready to go, we'll move on to the next steps in the staining protocol.

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Topics: Protocols, IHC, techniques

4 Steps to Better Immunohistochemistry: Step 1 - Antigen Retrieval


Posted by Carolyn P on Jul 8, 2015 7:00:00 AM

It’s Friday night - you’re tucked away in a dark, little room filled with microscopes.You could be out with your friends right now, but you begged off because you were certain this immunohistochemistry was going to reveal some small - but important - mystery of the universe to you.

Instead, you’re sitting here, cursing said universe and all its inhabitants, because all you see when you stare down the barrel of the scope is some indistinct fuzziness. And did the controls work - meh - who’s to say? There’s no sugar coating it – it’s a fail.

So, what’s next? If you’re like me, you’ll wing the slide into the trash with as much gusto as you can muster and head out to find your friends. Tomorrow, you’ll re-evaluate the experiment.

But where do you start? You probably know that a highly specific, high-affinity primary antibody is key to a successful IHC. But did you know that the companion reagents (i.e., buffers, etc.), which establish the pH and ionic strength of the system, are just as important? These reagents can influence the binding of the primary antibody to its epitope and dramatically affect the outcome of the assay.

To help you pick the best reagents for your assay (and make sure those Friday nights in the lab are worth the effort) we will spend the next several posts reviewing how companion reagents affect IHC. And, as an example we will describe our experience optimizing the protocol for one of our antibodies, PLK1 (208G4) Rabbit mAb.

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Topics: Protocols, IHC, Companion Reagents, techniques

Targeting Cancer Pathways - Tumor Metabolism and Proliferation


Posted by Carolyn P on Jun 24, 2015 8:00:00 AM


Cell Signaling Technology is proud to partner with the Koch Institute at MIT, Science, and Science Signaling to present the Targeting Cancer Pathways webinar series. These webinars will bring together thought leaders from around the world to share current findings and further cancer research community collaboration.

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Topics: Cancer Research, Webinars

4 Steps to Better ChIP results - Step 4 - Downstream Analysis


Posted by Carolyn P on Mar 11, 2015 3:00:00 AM

Part one of this series described the importance of including proper controls to your protocols. In Part 2 we discussed how chromatin preparation affects the final outcome of the experiment. Then we spent a post considering the immunoprecipitating antibody - and now let's analyze the data.

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Topics: Protocols, ChIP, techniques

4 Steps to Better ChIP results - Step 3- Choosing an Antibody


Posted by Carolyn P on Mar 4, 2015 3:00:00 AM

Part one of this series described the importance of including proper controls to your protocols. In Part 2 we discussed how chromatin preparation affects the final outcome of the experiment. Now, let's take a minute to consider the immunoprecipitating antibody.

Antibodies that are not highly specific to the target of interest may bind unpredictably and increase the background noise; and this may make it more difficult to detect less abundant or lower stability interactions.

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Topics: Protocols, ChIP, techniques

4 Steps to Better ChIP results - Step 2- Prep well


Posted by Carolyn P on Feb 25, 2015 3:00:00 AM

Part one of this series described the importance of including proper controls to your protocols. Now, we will examine how chromatin preparation affects the final outcome of the experiment.

First consider the type of interaction you are trying to detect:

  • High-frequency, very stable protein-DNA interactions like those between histones and DNA, occur frequently enough that they may still be detected even if the protocol is not fully optimized.
  • Low-frequency, less stable interactions like the binding of polycomb group proteins to specific genes (e.g., Ezh2), may fall under the detection threshold if the protocol fails to safeguard the integrity of the protein and the DNA, or if it relies on an antibody that is not highly specific to the target of interest.

Now it's time to consider your options for preparing your chromatin...

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Topics: Protocols, ChIP, techniques

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