Monday, May 03, 2004
Description Weskamp, G., and Otten, U. (1987). An enzyme-linked immunoassay for nerve growth factor (NGF): A tool for studying regulatory mechanisms involved in ngf production in brain and in peripheral tissues. Journal of Neurochemistry, 48 (6),1779-1786. Procedure 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 祃 of (GIG, 77 mg/ml) and 10.0 祃 (GAM, 66 mg/ml) to two separate tubes containing 10 ml COATING BUFFER. To a 96 well plate (Nunc), multipipet 100 祃/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癈 overnight. DAY 2 PROCEDURE BLOCKING Block the plates by applying 200 祃/well BLOCKING BUFFER to the freshly washed plates. Incubate at 4癈 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 祃 per sample is the minimum amount needed to obtain duplicate values (100 祃 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 祃 of 0.41 mg/ml NGF Ngf Standards I Tube # NGF 0.05% Acetic Acid Final Conc. 1 25 祃 975 祃 10 礸/ml 2 100 祃 #1 9.9 ml 100 ng/ml 2.5 100 祃 #2 900 祃 10 ng/ml 3 100 祃 #2 9.9 ml 1 ng/ml Use the above solutions to make the following more dilute standards Ngf Standards II Pg NGF/100 祃 Add Sample Buffer +0.1%tw 100 30 祃 of #2 3 ml 50 15 祃 of #2 3 ml 25 75 祃 of #2.5 3 ml 12.5 37.5 祃 of #2.5 3 ml 6.25 18.75 祃 of #2.5 3 ml 3.12 93.6 祃 of #3 3 ml 1.56 46.8 祃 of #3 3 ml PROTEIN RECOVERY Is determined by adding a known amount of NGF (spiking) to an adjacent aliquot of the sample being measured. The percentage recovery of NGF can then be determined by subtracting the pg of NGF per well of the sample from that of the spiked sample and then dividing by the added amount. A common spiking amount is 25 pg per well or 100 祃 sample (2.5 祃 of tube 2.5 to each 100 祃 of sample). DESIGNING THE PLATE To account for inter-plate variations, a complete set of standards should be included on each plate. To account for intra-plate variation, the standards should be alternated from top to bottom and from left to right within the plate. A common plate design will contain the 100 pg/well standard in the top left corner and the bottom left corner (A1, B1, G1, H1), the 50 pg/well standard in the top right and bottom right corner (A12, B12, G12, H12), the 25 pg/well standard next to the 100 pg standard (A2, B2, G2, H2), and the 12.5 pg/well standard next to the 50 pg standard (A11, B11, G11, H11). The lower concentrations are run in quadruplicate since more values fall into this range. The 6.25 pg/well standard placed in wells A3 through H3, the 3.12 pg/well standard in wells A10 through H10, the 1.56 pg/well standard in wells A4 through H4, and the blank or sample buffer (0 pg/well) in wells A7 through H7. The 12 samples are placed in the remaining 4-well pockets consisting of 2 GIG wells and 2 GAM wells. APPLYING THE STANDARDS AND SAMPLES Add 100 祃/well of each solution and incubate at 4癈 on the rotator overnight. DAY 3 PROCEDURE APPLYING THE MONOCLONAL Single wash, then triple wash the plates as described previously with special attention to the aspiration step. Maintaining the GIG then GAM sequence, aspirate the standards from low concentration (blank) to high concentration (100 pg). In this way, only 2 pipets are needed for the standards. Using a new pipet for each sample (12 pipets total), aspirate by removing solution from both GIG wells then the GAM wells. Once the plate has been washed, multipipet 100 祃/well (1:200) monoclonal anti-NGF antibody (Hazleton) prepared by adding 100 祃 IG3 (2.42 mg/ml) to 20 ml blocking buffer with the addition of 200 祃 of 10% Tween. Incubate the plates at 4癈 on the rotator overnight. DAY 4 PROCEDURE APPLYING SECONDARY ANTIBODY To a freshly washed plate, apply 100 祃/well of 1:625 Biotinylated Goat anti-rat IgG prepared by adding 32 祃 IgG (Calbiochem #60653) to 20 ml blocking buffer with the addition of 200 祃 of 10% Tween. Return plate to the 4癈 on the rotator for 6 hours. APPLYING STREPTAVIDIN After 6 hours, add 100 祃/well of 1:2000 streptavidin solution prepared by adding 10 祃 peroxidase conjugated streptavidin (Zymed #43-4323) to 20 ml blocking buffer. Incubate for 1 hour at room temperature on a rotator. CHROMAGEN DEVELOPMENT 10 min before washing the plates, add a 10 mg OPD (Sigma #P8287) tablet to 25 ml SUBSTRATE BUFFER and store tube in a dark drawer. Immediately before applying the chromagen to the plate, add 10 祃 of 30% H2O2 to the substrate buffer-OPD solution. Mix well and add 100 祃 to each well as quickly as possible. Incubate plate for 10 minutes on the room temperature rotator under a light proof object, such as an ice bucket lid. Store the remaining solution in a dark drawer to use on the second plate. READING THE PLATE Read the plate using a 492 nm wavelength in the plate reader (Bio Rad model 2550) interfaced with a Macintosh computer running MacReader 2.0 software. Before reading the plate, blank the reader by advancing to a column on a separate plate to which 100 祃 substrate buffer and 50 祃 of 2.5M Sulfuric Acid has been added. When the 10 minutes are up, add 50 祃/well of 2.5M Sulfuric Acid to stop the reaction and immediately read the plate. The colored end-product should be more stable and can be stored at 4癈 for extended storage. Recipes SOLUTIONS PBS SOLUTION, 50 mM Sodium Phosphate, Monobasic 1.50 g Sodium Phosphate, Dibasic 6.53 g Sodium Chloride 116.38 g Distilled H2O up to 1.0 liter PBS SOLUTION,10 mM, pH=7.4 50 mM PBS 1 liters Distilled H2O 4 liters COATING BUFFER pH=9.6 Sodium Azide .2 g Sodium Bicarbonate 2.93 g Sodium Carbonate 1.71 g Distilled H2O up to 1 liter WASHING BUFFER 10% Tween 20 ml 10 mM PBS 4 liters BLOCKING BUFFER 10 mM PBS 99 ml FCS 1 ml SAMPLE BUFFER 0.5% BSA (Albumin Bovine) 2.5 g .0001% or 1 礸/ml Aprotinin .0005 g 0.1 mM Benzethonium Chloride .022 g 0.1 mM PMSF (Phenylmethyl Sulfonyl Fluoride) .0086 g dissovled in 400 祃 95% ethanol 10 mM PBS up to 500 ml ACETIC ACID, 0.05% Glacial Acetic Acid .5 ml Distilled H2O 1 liter SUBSTRATE BUFFER, pH=5.0 Sodium Phosphate, dibasic 25.7 ml (2.84 g/100 ml=.2 M) Citric Acid 24.3 ml (1.92 g/100 ml=.1M) Distilled H2O up to 100 ml SULFURIC ACID, 2.5 M Sulfuric Acid 137.5 ml Distilled H2O up to 1000 ml REAGENTS Chemical Catalogue # Vendor NaH2PO4 (Anh.) S-0751 Sigma NaCl S-9625 Sigma BSA A-7888 Sigma Aprotinin A-1153 Sigma PMSF P-7626 Sigma Benzethonium Chloride B-8879 Sigma 10% Tween 28320 Pierce NaN3 S-2002 Sigma NaHCO3 S-8875 Sigma Na2CO3 S-2127 Sigma FCS F-4884 Sigma Na2HPO4 S-0876 Sigma Citric Acid C-0759 Sigma H2O2 H-1009 Sigma OPD P-8287 Sigma Streptavidin 43-4323 Zymed Biotinylated Goat Anti-rat IgG 60653 Calbiochem Supplies Tips (责任编辑:泉水) |