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A. DAY 1 PROCEDURE ??ABSORPTION OF THE POLYCLONAL AND PREIMMUNE SERUM The Goat Anti-Mouse (GAM) NGF polyclonal antibody recognizes 2.5B NGF and the Goat IgG (GIG) preimmune serum serves as a control for the antibody (Hazleton). The relative signal of a standard or sample is calculated by subtracting the GIG value from the GAM value for each sample. Add 8.6 μl of (GIG, 77 mg/ml) and 10.0 μl (GAM, 66 mg/ml) to two separate tubes containing 10 ml COATING BUFFER. To a 96 well plate (Nunc), multipipet 100 μl/well of GIG into alternating rows (A, C, E, G) and GAM into the remaining rows (B, D, F, H). In this step, and following incubations, place a thin strip of parafilm over the groove where the lid and bottom of plate meet to prevent evaporation of solutions. Incubate plates on a rotator (4-8 rpm) at 4°C overnight. B. DAY 2 PROCEDURE ??BLOCKING Block the plates by applying 200 μl/well BLOCKING BUFFER to the freshly washed plates. Incubate at 4°C for a minimum of 1 hour. In this step and all additional washing steps, first remove the present solution with a pasteur pipet attached to a vacuum. Aspirate all GIG (A,C,E,G) then all GAM (B,D,F,H) rows, changing the pipet to avoid contamination. Then, fill each row to the top with the WASHING BUFFER using the Nunc washing apparatus. Aspirate with pasteur pipets as before. A triple wash is required, this time using the Nunc washer to fill and aspirate each row 3x simultaneously before moving on to the next row. Dry the wells by removing the remaining liquid with pasteur pipets as described previously, but do not leave dried wells for an extended period of time. ??SAMPLE PREPARATION A volume of 400 μl per sample is the minimum amount needed to obtain duplicate values (100 μl per well for 2 GIG and 2 GAM wells). 0.1% Tween is added to the SAMPLE BUFFER to increase the amount of NGF removed from the sample. Typically, a 10x dilution (w/v) of tissue samples will give a signal sufficient for detection. Once diluted, samples are homogenized on ice for 30 seconds using a cell sonicator at micro tip limit. Wash the tip between samples with distilled water. Centrifuge for 30 minutes at 15,000 G Force under refrigeration. ??PREPARATION OF NGF STANDARDS When preparing these standards, mix gently, the NGF is fragile and over mixing by vortexing can destroy the protein. Use 25 μl of 0.41 mg/ml NGF
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