It's time to check out another video from the CST Tech Tips playlist! In this edition of Tech Tips, we'll tackle a common protocol question customers ask our ChIP team: how much antibody to use for chromatin immunoprecipitaion (ChIP) experiments. Adding more antibody isn't always better - watch the video to learn why. 

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How much antibody should I use in ChIP assays? | CST Tech Tips

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Research trends in macrophage plasticity

It’s an exciting time for immuno-oncology research, as potential predictive biomarkers from an expanding collection of cell types are being pursued. Explore the plasticity of tumor-associated macrophages (TAMs) and challenges in distinguishing M1- versus M2- functional states in this 5-minute video featuring CST Developmental Scientist Sarah Klein, PhD. 

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So you've set the timer to five minutes for the first of three TBST washes for your western blot membrane. Now what? Sure, you could check your email or social media for the 30th time before lunch. Or you could do something informative, like check out a CST Tech Tips video! This is a new short video series featuring the same scientists who develop and validate CST antibodies, here to offer insights and protocol tips.

Each summer, college and high school interns at CST get a chance to sample work life in a biotech environment. They are typically in the early stages of considering potential career paths, so what better time to set up a Q&A panel? Five CST employees shared some of their career development experiences during a recent “lunch and learn” session with the interns. Selections from the session have been transcribed and edited for brevity. –ed.

Q: Which do you like better, being on the bench or managing?

You’re gathering data from all your experiments and preparing to present to your advisor and thesis committee at your annual progress report. You have an interesting hypothesis, and you have a validated antibody that recognizes your target protein on a western blot (WB). The molecular weight of the band is correct, and the expression of the target protein changes just the way you predicted it would. Now, you know — and you’d bet the house on it — when that powerpoint slide comes up, someone on your committee is going to ask about loading controls.

Early exploration of unmapped biological signaling pathways were carried out using radiolabeled phospho-imaging. The development of phospho-specific antibodies to detect and quantify protein phosphorylation made life easier for researchers (less ³²P waste to deal with), but the interpretation of data from these experiments comes with its own set of caveats. 

Part four of a series on immunofluorescence techniques. Check out previous posts on Validation, Experimental Controls, and Fixation/Permabilization. 

After your samples have been prepared, it's time to incubate them with a well-validated antibody,  the workhorse of immunofluorescence. If you are a seasoned pro at IF experiments, you are probably used to checking the antibody datasheet (or web page) for the recommended dilution. But have you ever wondered where those recommendations come from?

The Zika virus (ZIKV) is related to flaviviruses that cause dengue and yellow fever, and is spread via mosquito (Ae. aegypti and Ae. albopictus) bites and secondarily via sexual transmission. ZIKV infection in adults is associated with mild dengue-like disease or Guillain-Barré syndrome, an autoimmune disorder of the peripheral nervous system. Most alarmingly, ZIKV is also transmitted across the placental barrier during pregnancy and can lead to the severe birth defect microencephaly (underdevelopment of the brain).

Part Three of a four-part series on Immunofluorescence. Check out The Importance of Validation and Experimental Controls.

The performance of an antibody is a crucial determinant in getting reliable immunofluorescence (IF) results. Equally important is the preparation of the biological sample - cells or tissue used in your experiments - before any antibodies are introduced. The fixation and permeabilization of your samples are key steps that can determine your experiment’s failure or success. The ideal fixative preserves a “life-like” snapshot while quickly stopping the degradative process of autolysis by crosslinking and inhibiting endogenous enzymes. This post provides examples of how different antibodies perform at their best using different protocols.

 Part two of a four-part series on Immunofluorescence. Check out our posts on Validation and Fixation and Permeabilization. 

After months of hard work, your research has honed in on a hypothesis you can test with immunofluorescence (IF). You've chosen antibodies and performed pilot IF experiments (see The Importance of Validation), and the localization of the protein appears reasonable. But how can you be sure the IF data you've acquired represents real biological phenomena? We present two examples of experimental controls in this post.