Download PDF by Margaret M. Manson: Immunochemical Protocols (Methods in Molecular Biology Vol


By Margaret M. Manson

ISBN-10: 0896032043

ISBN-13: 9780896032040

Highlights contemporary advances in immunochemical strategies and their effect on uncomplicated learn and medical drugs, combining the forces of specialists in quite a lot of subspecialties to hide a wealth of knowledge.

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Additional info for Immunochemical Protocols (Methods in Molecular Biology Vol 10)

Sample text

Block excess glutaraldehyde by adding 100 PL of 1Mglycine and stirring for a further 15 min. Use directly or dialyze overnight against PBS and store at -2OOC. 2. PPD kit: Read the instructions supplied with the kit very carefully. Inhalation of the ether-dried tuberculin PPD is dangerous for tuberculinsensitive people to handle. Follow specific instructions to couple 2 mg of peptide to 10 mg of PPD and, after dialysis, store at -2OOC. 2. Antigens for Immunization 1. Suspend whole cells in PBS or DMEM at 5 x 106-10’ cells/ml, or 2.

6. Redissolve the precipitate in water and dialyze against PBS for immediate use or PB/lO (five changes of 10 vol) for fractionation by tonexchange chromatography. 2. Ion-Exchange Chromatography 1. Run the dialyzed 45% (NH,)$O, fraction onto a DE52 column equilibrated with PB/lO and elute with the same buffer. Most rat and mouse antibodies of the IgGz, and IgGsb subclasses do not bind and appear in the eluate. 2. 6 to elute IgGswith lower isoelectric points. 6 and B is the same buffer containing 1MNaCl.

7. After rocking for 1 h at room temperature, open the bags, dispose of the radioactive supernatant safely, then wash the blots four times with PBST and dry at 37OC. 8. Secure the blot strips to a sheet of Whatman 3MM paper using PhotomountTM or other spray-on adhesive, then autoradiograph at-70°C using prefogged X-ray film and an intensifying screen (4). 8. , Chapter 10) that is either soluble (plate assays) or insoluble (Western blots) (see Note 11). The protocols for the use of either radiolabeled or enzyme-conjugated antibodies are the same until the completion of MAbs to Recombinant 57 Proteins washing following treatment with the second antibody or, in the case of competitive assays, the specific antibody.

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Immunochemical Protocols (Methods in Molecular Biology Vol 10) by Margaret M. Manson

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