My lab is looking to use NIP 552 blocking reagent (GE Healthcare) outside of the kit it is a part of. Is there anyway to put the powder in solution to be used as a blocking reagent?
Dear Sir. Concerning your issue about the solubility of NIP 552 blocking reagent. Immunization with 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)- protein conjugates, and assay of antisera by binding of N131IP- E-amino-n-caproic acid (NM31IP-aminocap), is used to investigate the response of CBA mice. Primary immunization is obtained more efficiently with alum-precipitated conjugates mixed with pertussis, than with conjugates alone or in Freund's complete adjuvant, and is most efficient with conjugates of chicken serum globulin (NIP-CG) or edestin. Measurement of hapten binding capacity follows the simplified method previously described for albumin (Mitchison, 1964). Sera are stored at -2° at 1: 6 dilution (0.6 ml) in pH 8-4 borate buffer (Farr, 1958). Two dilutions in normal rabbit serum (10 per cent in pH 8f4 borate buffer) are made from each serum, to 1: 36 and 1: 180 (0f25 ml), by means of a metered pump and Lang-Levy pipette (0.05 ml). Into these, and into 0-25 ml of the original 1: 6 dilution, is pumped an equal volume of 2 x M-8 NM3IP-aminocap (or other radioactive hapten). After incubation overnight at 40, 0 5 ml saturated ammonium sulphate is added by pump, and the tubes shaken and centrifuged (3000 g, 10 minutes). The supernatants are then decanted and the precipitates counted, together with one set of control tubes containing the volume of N131IP-aminocap originally added (high control), and another set which contained 0-25 ml normal rabbit serum (10 per cent in pH 8-4 borate buffer) and which then receives N131IP-aminocap, etc., along with the experimental tubes (low control). Calculation of binding capacity makes use of the assumptions concerning binding that were given in Brownstone et al. (1966) (35 per cent binding, binding proportional to log (antibody concentration)). Values of binding greater than 70 per cent and less than 10 per cent are discarded in order to reduce experimental error, except for the 1: 6 dilution tube, where values are accepted down to 0-83 per cent binding (M-9 binding capacity). This exception allows the assay to be applied to weak sera. Values of binding capacity below this range are set arbitrarily at M-9, in order to permit calculation of a geometric group mean. The estimates obtained for a single antiserum, not normally more than two in number, are averaged, and the geometric mean and standard deviation for a group of antisera calculated. The vertical bars in the figures indicate the standard deviations obtained in this way. No standard deviation is given for groups which include sera with binding capacity
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