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

By Biji T. Kurien, R. Hal Scofield

This specified quantity offers tools and strategies for detection after blotting. Chapters consultant readers via a few adaptations at the topic of protein move to sturdy aid through detection, offering diversifications of conventional innovations, and unique equipment of protein blotting. Written for the Methods in Molecular Biology sequence, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and tips about troubleshooting and fending off identified pitfalls.

Practical and authoritative, Detection of Blotted Proteins: tools and Protocols provides a number of suggestions in keeping with the Western blot, supplying distinctive, with no trouble reproducible equipment, suggestions, and choices without delay and simply transferable to the laboratory environment.

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Additional info for Detection of Blotted Proteins: Methods and Protocols

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In acidic solutions the dye sticks to the amino groups of the proteins by electrostatic and hydrophobic interactions. CBB staining and its various modifications [6–9] are generally applied to acrylamide gels but they are applicable to PVDF or nitrocellulose membranes. 1 % Coomassie dye dissolved in 45 % (v/v) methanol, 45 % (v/v) water, and 10 % acetic acid. The background is destained with 25 % (v/v) methanol, 65 % (v/v) water, and 10 % acetic acid (see Notes 8 and 9). 1 Alternate Method to Stain with CBB This staining procedure is used when low background staining and a high degree of sensitivity are required.

For reproducible results it is preferable to keep volumes of samples below 2 µL. This ensures that the sample remains within the 1 cm2 boundary on the nitrocellulose. 13. For all the protein determination methods presented here, the standard curves of protein concentration proceed from the origin of the graph, provided that the correct “blank” or “control” has been used. References 1. Goldring JPD (2015) Spectrophotometric methods to determine protein concentration. In: Kurien BT, Scofield RH (eds) Western blotting, Methods in molecular biology.

J Biomed Biotechnol 2011:245291 Jennings A (1999) Lyophilization: introduction and basic principles. Taylor & Francis, Boca Raton Roy I, Gupta MN (2004) Freeze-drying of proteins: some emerging concerns. Biotechnol Appl Biochem 39:165–177 Schade R, Behn I, Erhard M, Hlinak A, Staak C (2001) Chicken egg yolk antibodies, production and application. Springer lab manual. Springer, Berlin Ning Z, Hawley B, Seebun D, Figeys D (2014) Apols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

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