Webinar - Multiplex Immunohistochemistry (IHC)


Posted by Liana G on Jan 13, 2016 3:00:00 AM

Analysis of Immune Checkpoint Control Protein Co-expression in Breast and Ovarian Cancer Using Novel Rabbit Monoclonal Antibodies and Multiplex IHC

With an increasing number of biomarkers and, often, limited availability of biopsy material, there is a growing need for multiplexed assays for both research and clinical purposes. IHC based solutions are particularly attractive in the field of immuno-oncology, as maintaining spatial context within the tumor microenvironment provides meaningful and potentially actionable information.

Watch the video below.

 

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Topics: Protocols, IHC, Cancer Immunology, Cancer Research, mIHC

Webinar - Simplify Proteomics


Posted by Claire S on Dec 16, 2015 3:00:00 AM

Post Translational Modification: Antibody Enrichment for Mass Spectrometry-based Proteomics

Continuing our theme of simplifying proteomics we present a webinar featuring Matthew Stokes, Ph.D., principal scientist of the proteomics group here at Cell Signaling Technology and Christopher Rose, Ph.D., a postdoctoral research fellow in the Gygi Lab at Harvard Medical School.

Dr. Stokes describes PTMScan® technology, a method that uses antibodies to enrich specific post-translationally modified (PTM) peptides (e.g., phosphorylated, methylated, ubiquitinated etc) from a complex mixture prior to LC-MS/MS analysis. Dr. Rose demonstrates how, by combining PTMScan technology with isobaric labeling, specifically with tandem mass tags (TMTs), his lab quantified over 15,000 ubiquitination events in Bortezomib treated cells.

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Topics: Protocols, Cancer Research, Proteomics, PTMScan

5 Steps to Publication-Worthy IF Images


Posted by Tara W on Sep 23, 2015 8:00:00 AM

A picture is worth a thousand words, or in the case of immunofluorescent imaging, a thousand proteins. The images used to illustrate a scientific experiment should convey as much information as the text itself. Here at CST, we pride ourselves in the quality of our antibodies and our rigorous validation process. When we approve our primary antibodies for IF, we like to showcase them using high quality images generated in-house. Beyond our recommended IF protocols (check it out here), here are some additional considerations to make when planning your IF staining. 

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Topics: Protocols, Antibody Performance, Antibody Validation, IF-IC

Intracellular Flow Cytometry in Action


Posted by Liana G on Aug 12, 2015 8:00:00 AM


Traditionally, flow cytometry has been used to identify distinct cell types within a heterogeneous pool of cells, based on extracellular or surface marker expression, an application commonly known as immuno-phenotyping. However, this technology is also readily amenable to intracellular target detection and can be successfully applied to the study of complex signaling events.

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Topics: Protocols, Flow, Cell Biology, techniques

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

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

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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