Immunohistochemistry (the localization of proteins in a tissue by binding antibodies to specific antigens) is a technique where one protocol definitely does not fit all. Each model organism seems to have its own quirks, whether it be in the fixative used, the methods needed for antibody penetration, issues with autofluorescence or even just figuring out which cross-species antibodies work in a given system. To that end, we’ve been working on expanding our coverage of immunohistological protocols. The February issue of CSH Protocols brings methods for plant sections, using both avidin-biotin and alk-phos, as well as a method for whole-mount immunocytochemistry in Xenopus embryos from John Wallingford’s lab at the University of Texas (they provided the lovely cover image for this month).

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Our protocol on Preparation of GST Fusion Proteins has seen a great deal of activity on the site, so this month we’ve supplemented it with several other protocols and background articles. This should give a better set of tools to do GST pull-downs and Far Westerns. Read the rest of this entry »

Older methods of assaying for protein:protein interactions in situ relied upon chemical cross-linking and antibodies. The obvious problem with these methods is that they alter the physiological conditions within the cell. Plus, they offer limited information on spatial distribution of the proteins in question. These problems can be solved by instead employing Fluorescence Resonance Energy Transfer (FRET) based techniques. Read the rest of this entry »

CSH Protocols’ April Issue features a set of protocols from Michael Weber and colleagues at the University of Virginia detailing methods for using genetically engineered kinases to screen for novel protein kinase substrates. Determining the direct substrate of a protein kinase is important for understanding its function in vivo. Read the rest of this entry »

To analyze highly complex mixtures of proteins, a combination of 2D-PAGE and Mass Spectrometry (MS) is usually used. However, this can be problematic when dealing with low abundance proteins, membrane-associated or bound proteins, or proteins with extremes in pI or molecular mass. Read the rest of this entry »