MATERIALS PROVIDED Bring all reagents to room temperature before use. GENERAL EL
ID: 131965 • Letter: M
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MATERIALS PROVIDED Bring all reagents to room temperature before use. GENERAL ELISA PROTOCOL Plate Preparation 1. Dilute the Capture Antibody to the wo Capture Antibody (Part 840143, 1 vial) . 144 ?g/mL of goat anti-mouse TNF-a when reconstituted with 1.0 mL of PBS. After reconstitution, store at 2-8 C for Up to 60 days or aliquot and store at-2 Cto-70° C in a manual defrost freezer for up to 6 months. "Dilute to a working concentration of 0.8 ?g/mL in PBS. without carrier protein. concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 HL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature. 2. Aspirate each well and wash with Wash Buffer the process two times for a total of three Detection Antibody (Part 840144, 1 vial) - 72 ug/mL of biotinylated goat anti-mouse TNF-a when reconstituted with 1.0 mL of Reagent Diluent (see Solutions Required section). After reconstitution, store at 2-8° C for up to 60 days or aliquot and store at 20° C to -70 C in a manual defrost freezer for up to washes. Wash by filling each well with Wash Buffer (400 ?L) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by or by inverting the plate and blotting it against n paper towels. 6 months. Dilute to a working concentration of 400 ng/mL in Reagent Diluent. 3. Block plates by adding 300 uL of Reagent Diluent well. Incubate at room temperature for a minimum of 1 hour Standard (Part 840145, 1 vial) - 280 ng/mL of recombinant mouse TNF-a when reconstituted with 0.5 mL of Reagent Diluent (see Solutions Required section). Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Store reconstituted standard at 2-8° C for up to 60 days or aliquot and store at -70° C for up to 6 months. A seven point standard curve using 2-fold serial dilutions in Reagent Diluent, and a high standard of 2000 pg/mL is recommended. 4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition. 1. Add 100 ?L of sample or standard in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room 2 Prepear tion spraton/wash as n step 2 of Plate 3. Add 100 uL of the Detection Antibody, diluted in to each well. Cover with a new Streptavidin-HRP (Part 890803, 1 vial) -1.0 mL of streptavidin conjugated to horseradish-peroxidase Store at 2-8 C for up to 6 months after initial use. DO NOT FREEZE. Dilute to the working concentration Reagent dhesive strip and incubate 2 hours at room temperature. Repeat the aspiration/wash as in step 2 of Plate Preparation. Add 100 uL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light Solutions Required section). 5. SOLUTIONS REQUIRED PBS 137 mM NaCI, 2.7 mM KCI, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 72-74, 0.2 ?m filtered. 6. Repeat the aspiration/wash as in step 2. 7, Add 100 ?L of Substrate Solution to each well. Wash Buffer-0.05% Tween. 20 in PBS, pH 7.2-7.4 (R&D; Systems Catalog # WA126). Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light B. Add 50 ??of Stop Solutionto each well. Gently tap Reagent Diluent'-1 0.2 ?m filtered (R&D; Systems Catalog # DY995) Quality of BSA is critical (see Technical Hints). the plate to ensure thorough mixing. Determine the optical 9. of each well a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate. Substrate Solution-1:1 mixture of Color Reagent A (H202) and Color Reagent B (Tetramethylbenzidine) (R&D; Systems Catalog # DY999). Stop Solution-2 N H2S04 (R&D; Systems Catalog # DY994)Explanation / Answer
Your provided data: Number of sample 35 and 3 repeats and 100 micro litres in each well = 35x3x100 = 10500 micro litres = 10500/1000 = 10.5ml
1% BSA means 1g in 100ml PBS but you require 10.5ml
That is (10.5/100)1g = 0.105g or 105mg of BSA dissolve in 10.5ml of PBS (first dissolve in lets say 5 ml of PBS and make up the volume to 10.5ml dont add directly in to 10.5ml PBS, always makeup the solutions)
In summary
BSA required 105mg (0.105g)
PBS required 10.5ml
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