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The assay for cGMP was carried out using a commercially available radioimmunoassay kit, the 'Cyclic GMP assay system' (Amersham). The assay is based on the competition between unlabelled cGMP and a fixed quantity of [^H]-cGMP for binding to a specific antibody which has a high specificity and affinity for cGMP. All solutions were made up as specified in the assay protocol supplied with the kit. Standard concentrations of unlabelled cGMP (0.25-8pmol per lOOpl) were prepared in 50mM Tris-HCl buffer containing 4mM EDTA (4®C, pH 7.5) (cGMP assay buffer). For the cerebellar slice samples, the assay was set up on ice in Eppendorf tubes. To these tubes were added either lOOpl o f standard unlabelled cGMP, lOOpl of unknown sample (which was diluted in cGMP assay buffer if the amount of cGMP was too high for the standard range), lOOpl of cGMP assay buffer for zero-dose binding or lOOpl of the supplied 'blank' reagent for the determination of the assay blank. For the forebrain slice samples, the assay was set up on ice in Eppendorf tubes. To these were added either lOOpl of standard unlabelled cGMP plus lOOpl of cGMP assay buffer, 200pl of unknown sample (more sample was required because the levels were low), 200pl of cGMP assay buffer for zero-dose binding or lOOpl of the supplied 'blank' reagent plus lOOpl of cGMP assay buffer for the determination of the assay blank. Zero-dose, blank and standard concentrations of unlabelled cGMP were assayed in duplicate, and unknown samples were assayed singly. A [^H]-cGMP

Chapter 6 : GABAg receptors and cGMP levels in the cerebellum and forebrain (approximate S.A. = 20Ci/mmol) solution was made up using distilled HgO and 50pl (0.4pmol, approximately 8nCi) aliquots dispensed into each assay tube. 50pl aliquots of antibody solution (approximately 35% of [^ -c G M P bound at zero-dose) made up in distilled HjO were then added to every tube. Total assay volumes were 200pl for the cerebellar slice experiments and 300pl for the forebrain slice experiments. The tubes were vortex mixed, placed in an ice bath and incubated for 90 min at 4®C.

The bound [^H]-cGMP was separated from the free [^H]-cGMP by adding 1ml aliquots o f cold ammonium sulphate (60% saturated, 4®C) to each tube. The tubes were vortex mixed, placed back in the ice bath, then left to stand for 5 min (timed from the addition o f ammonium sulphate to the last assay tube). 5 min after the addition of ammonium sulphate, the assay tubes were centrifuged for 5 min at 11000 rpm (4®C) in an Eppendorf bench top centrifuge. The supernatant was decanted from the assay tubes and the tubes placed upside down on tissue to drain. Excess liquid was carefully wiped from the neck of each tube, taking care not to disturb the precipitate. Aliquots (1.1ml) of distilled HgO were then added to each tube and vortex mixed until the precipitate dissolved. 1ml samples were then removed from each tube and mixed with 10 ml of Optiphase "Safe" Scintillation Fluid and left overnight. The amount of radioactivity was then quantified by liquid scintillation spectophotometry for a 4 min count in an LKB 1219 Rackbeta liquid scintillation counter. On the advice of the supplied assay protocol, counts per minute (cpm) were used directly for calculations.

From the data, the amount of bound ligand at zero dose (cpm) was calculated, the blank (cpm) subtracted and the value designated The amount of bound ligand in the presence of standard amounts of cGMP (cpm) and in the unknown samples (cpm) was calculated, the blank (cpm) subtracted and these values designated C,. Graphs were then plotted of e y e , against pmol of cGMP in the standards (0.25 - 8pmol) for the cerebellum (total volume 200pl) (Figure 6.2.) and forebrain (total volume 300pl) (Figure 6.3.) protocols. A straight line could be obtained with an intercept of 1.0 on the ordinate (at zero-dose). From the e y e , values for the unknown samples, the amount of cGMP (pmol per assay tube) could then be determined from the equation of the line. Ultimately the results were expressed as pmol/mg of protein after the protein content of the slices was calculated.

9.0 .005 0.858 7.5 6.0 X Ü '— 4.5 o Ü 3.0 1.5 0.0 4 6 8 0 2 cGMP (pmol)

Figure 6.2. Standard curve for the assay of cGMP in cerebellar slices

G r^ h represents concentration o f unlabelled cGMP standards (0.25 - Spmol per lOOpl) against binding to cGMP antisCTum of pH]-cGMP (0.4pmol, approximately 8nCi per SOpl). The amount o f bound ligand at zero- dose (cpm ) was calculated, the blank (cpm) subtracted and the value designated The amount o f bound ligand in the presence o f standard amounts o f unlabelled cGMP (cpm) was calculated, the blank (cpm) subtracted and these values designated C,. The graph was then plotted o f C /C , against pmol o f unlabelled cGMP in the assay standards for the cerd^ellar (total volume 200pl) protocol. A straight line could be obtained with an intercq)t of 1.0 on the (xdinate (at zero-dose). Zero-dose, standard concentrations o f unlabelled cGMP and blank were assayed in diplicate and the graph represents a single typical experiment.

Chapter 6 : GABA„ receptors and cGMP levels in the cerebellum and forebrain X U o O 9.0 .0 3 0 0 .8 8 0 7.5 6.0 4.5 3.0 1.5 0.0 2 4 6 8 0 cQMP (pmol)

Figure 6.3. Standard curve for the assay o f cGMP in forebrain slices

Graph represents CŒicentration o f unlabelled cGMP standards (0.25 - Spmol per l(X)pl) against binding to cGMP antiserum of pH]-cGMP (0.4pmol, approximately SnCi per 50pl). The amount o f bound ligand at zero- dose (cpm) was calculated, the blank (cpm) subtracted and the value designated C„. The amount o f bound ligand in the presence o f standard amounts o f unlabelled cGMP (cpm) was calculated, the blank (cpm) subtracted and these values designated C,. The graph was then plottW o f C /C , against pmol o f unlabelled cGMP in the assay standards for the forebrain (total volume 300pl) protocol. A straight line could be obtained widi an intercept o f 1.0 on the adinate (at zero-dose). 2Lero-dose, standard concentrations o f unlabelled cGMP and blank were assayed in diq)licate and the graph represents a single typical experiment.