By John Pound

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3. Careful attention should be paid to temperature during preparation of the antigen;always work between 2 and 4°C. Be particularly careful during sonication; wait 2-3 min between consecutive sonications. 4. This is a crucial point in the procedure. If too much PBS is added, the pieces of nitrocellulose will swirl around the probe and disintegration does not occur. 4 cm2 of nitrocellulose). For these quantities, one or two 10-s cycles suffice to get powdered nitrocellulose. We mention the volume as a reference since the surface of nitrocellulose-bound antigen may vary.

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Such emulsions are thick, do not separate even after standing for long periods, and do not disperse if pipeted onto the surface of water. Immunogens administered in Freund's adjuvant can persist for weeks, and there is thus no point in repeating From: Methods in Molecular Biology, Vol. 80: Immunochemical Protocols, 2nd ed. Edited by: J. D. , Totowa, NJ Page 16 immunizations too frequently. Freund's complete adjuvant (FCA), which contains killed pertussis bacteria to induce massive nonspecific inflammation, causes ulceration (resulting in loss ofimmunogen and considerable discomfort to the animal) if administered in too large a bolus in one place or if given more than once.

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Immunochemical Protocols (Methods in Molecular Biology) by John Pound

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