Glucosidase Alpha, Acid (GaA) BioAssay(TM) ELISA Kit (Human)

The Human Glucosidase alpha, Acid (GaA) ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of GaA in human tissue homogenates, cell lysates and other biological fluids. Detection Range: 0.156-10ng/ml, Sensitivity: 0.054ng/ml, Precision: Intra-Assay: CV<10%, Inter-Assay: CV<12%, Kit Components: *025324A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use., *025324B: Standard, 2x1vial , 025324C: Standard Diluent, 1x20ml, *025324D: Detection Reagent A, 1x120ul , *025324E: Detection Reagent B, 1x120ul , 025324F: Assay Diluent A, 1x12ml, 025324G: Assay Diluent B, 1x12ml, 025324H: TMB Substrate, 1x9ml, 025324K: Stop Solution, 1x6ml, 025324L: Wash Buffer, 30X, 1x20ml , Storage and Stability: Store *025324A, *025324B, *025324D and *025324E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap. Materials Required But Not Supplied: 1. Microplate reader with 450 ± 10nm filter., 2. Precision single or multi-channel pipettes and disposable tips., 3. Eppendorf Tubes for diluting samples., 4. Deionized or distilled water., 5. Absorbent paper for blotting the microtiter plate., 6. Container for Wash Solution, Sample Preparation and Storage: Tissue Homogenates:, The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed thoroughly in ice-cold PBS (0.02M pH 7.0-7.2) to remove excess blood and weighed before homogenization. The tissues were minced to small pieces and homogenized in 5-10ml of PBS with a glass homogenizer on ice (Micro Tissue Grinders works too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifuged for 5 minutes at 2500×g. The supernatant was removed and assayed immediately or aliquoted and stored at -20°C or lower. Cell Lysates: Cells must be lysed before assaying according to the following directions. , 1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). , 2. Wash cells three times in cold PBS. , 3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4X (or freeze cells at -20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3X.) , 4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris. Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles. Assay Procedure Summary: 1. Prepare all reagents, samples and standards., 2. Add 100ul standard or sample to each well. Incubate 2 hours at 37°C., 3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C., 4. Aspirate and wash 3 times., 5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C., 6. Aspirate and wash 5 times., 7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37°C., 8. Add 50ul Stop Solution. Read at 450nm immediately., ,