By Iain J. McEwan (auth.), Iain J. McEwan (eds.)
Acting largely to regulate styles of gene expression, nuclear receptors play very important roles in the course of embryonic improvement and within the rules of metabolic and reproductive capabilities in grownup lifestyles, which proves this superfamily of ligand-activated transcription elements to be a vital a part of organic existence. In The Nuclear Receptor Superfamily: equipment and Protocols, specialist researchers describe various molecular, structural and mobile organic concepts presently used to enquire the structure-function of nuclear receptors, including experimental methods which can bring about new healing techniques for treating nuclear receptor-associated illnesses. Written within the hugely winning Methods in Molecular Biology™ sequence structure, the chapters during this quantity include short introductions to the subjects, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, in addition to notes from the specialists to focus on tips about troubleshooting and heading off recognized pitfalls.
Cutting-edge and simple to take advantage of, The Nuclear Receptor Superfamily: tools and Protocols offers helpful and time-saving tips for all these project learn during this ever-growing box of study.
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Extra info for The Nuclear Receptor Superfamily: Methods and Protocols
Centrifuge tubes for 5 min (900 × g) at 4°C to pellet cells. Remove supernatant by decanting. Loosen cell pellet by gently vortexing to prepare for intracellular labeling of steroid hormone receptors. 14. Using separate pipette tips, add 10 µl of rat serum to each tube. Serum provides a blocking mechanism that will inhibit nonspecific binding of antibodies to other intracellular proteins. Mix serum with cells by thoroughly pipetting up and down. Place tubes on ice for 10 min. 15. Using separate pipette tips, add 10 µl of purified isotype control (mouse IgG, rabbit IgG) or anti-rat steroid hormone receptor (PR, GR, AR) antibody to appropriate tube (see Note 10).
If necessary, digestion with DNase I, collagenase, and hyaluronidase (all from Sigma) for at least 1 h at 37°C may be necessary to further break up tissue. Prior to enzymatic digestion, centrifuge sample for 5 min (900 × g) at 25°C to pellet cells. Remove supernatant by decanting, and resuspend pellet in enzymes to ensure maximal digestion. After digestion step, add 5 mL of conditioned medium to each tube to neutralize enzyme activity. 3. Following dissection and digestion of tissue or collection of cells from culturing conditions, it is important to filter sample to ensure a uniformly single-cell suspension and to remove any clumps or tissue debris that could potentially clog the flow cytometer.
PR expression is compared with the isotype control antibody (Mouse IgG), which provides information on background measure of fluorescence. 87 Fig. 4. Statistical information automatically generated by flow cytometry software provides different information about cells. In this example, quadrant statistics is provided for Fig. 2b. Quadrant statistics gives information generated from dot-plots, including percentiles for each quadrant (representing positive and negative expression of parameters); mean fluorescence intensity (representing average amount of receptor expressed by individual cells); and raw numbers of events (cells) collected for each quadrant.
The Nuclear Receptor Superfamily: Methods and Protocols by Iain J. McEwan (auth.), Iain J. McEwan (eds.)