Download Detection of Blotted Proteins: Methods and Protocols by Biji T. Kurien, R. Hal Scofield PDF

By Biji T. Kurien, R. Hal Scofield

This distinct quantity offers tools and methods for detection after blotting. Chapters advisor readers via a few adaptations at the topic of protein move to strong help by way of detection, providing diversifications of conventional thoughts, and unique equipment of protein blotting. Written for the Methods in Molecular Biology sequence, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and pointers on troubleshooting and heading off identified pitfalls.

Practical and authoritative, Detection of Blotted Proteins: equipment and Protocols offers various innovations in keeping with the Western blot, offering special, without problems reproducible equipment, information, and choices at once and simply transferable to the laboratory atmosphere.

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Additional resources for Detection of Blotted Proteins: Methods and Protocols

Example text

E) Stock solution stable for over a year. 2. Disadvantages of Ponceau S Staining. (a) Staining is not permanent. (b) Staining is not very sensitive. (c) Difficult to record photographically. 3. Ponceau S is kind of an odd stain in that in order to visualize the staining, one needs to briefly stain (5–10 min) and then very briefly wash with water (just until a little of the red comes off)—5–10 s at a time, discard the water and look, add more water to clear the background a bit, then swish around and discard the water.

Blocking agents are a critical component for western blotting protocols as these diminish nonspecific binding by blocking off-target sites on the membrane. A variety of blocking agents are available and these are selected in an empirical manner, as no single blocker is compatible with every system. The best blocking agent and method for any particular assay will be an optimized but not absolute choice. Here, we describe characteristics of the most common blocking agents used in western blotting and discuss their advantages and disadvantages.

2. Decant the stain, and rinse the membrane several times with distilled water (D/W) until the protein bands are visible. 3. Use a soft lead pencil to mark the major protein spots and molecular-weight markers even when the membrane is wet. 4. Continue rinsing the membrane with D/W with gentle agitation until the Ponceau S is removed. 2 Staining Blots with India Ink 37 1. 3 %), with two changes at 5 min each. 2. Place the blot in ~100 mL of India ink suspension. 3. Incubate at room temperature (RT) for 15 min to 18 h.

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