Sample preparation for ELISA

Although the preparation of samples for ELISA must always be optimized based on the target molecule and the specific assay at hand, there are general guidelines for the correct preparation of the samples before starting the experiment. In this post, we bring you a general guide for the preparation of samples for ELISA, including those that are used most frequently in these immunoassays. To know more visit MMP-9 ELISA Kit.

GENERAL TIPS FOR SAMPLE PREPARATION FOR ELISA

As a general rule, serum, plasma, cell and tissue extracts are used diluted 50% with the buffer.rnThe protein concentration in the extracts should be at least 1 mg / mL.rnThe conservation of the samples will be done based on the expected storage period: Refrigerated (4-8ºC) for periods of less than 48 hours, frozen (-20ºC) for periods of less than a month and deep-frozen (-80ºC) for periods of up to 6 months. PREPARATION OF SAMPLES FOR ELISA DEPENDING ON THE TYPE OF SAMPLE1.- Culture supernatant Centrifuge the cell medium for 10 minutes (1500 rpm, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 2.- Cell lysates Wash the cells with PBS.rnHomogenize and lyse the cells by adding the lysate buffer.rnCentrifuge the lysate for 5 minutes (10000g, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 3.- Extracts of tissue Wash the tissue with PBS.rnCut into 1-2mm sections and keep them on ice.rnPrepare the extraction buffer (can be previously prepared and stored at 4ºC)rnAdd 300uL of this extraction buffer for every 5mg of tissue and homogenize.rnShake the sample for 1 hour at 4ºC.rnCentrifuge for 5 minutes (10000g, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 4.-Serum Collect the blood in a tube without additives, and keep it at room temperature for 30 minutes.rnCentrifuge for 10 minutes (3000rpm, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 5.- Plasma Collect the blood in a tube with anticoagulant, and keep it at room temperature for 30 minutes.rnCentrifuge for 10 minutes (3000rpm, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 6.- Urine Collect urine in a sterile container.rnCentrifuge the sample for 1 minute (10000g, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 7.- Saliva Collect saliva with a suitable device.rnCentrifuge for 2 minutes (10000g, 4ºC).rnUse immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze/thaw cycles. 8.- Milk Centrifuge the sample for 15 minutes (1500g, 4ºC).rnCollect the aqueous fraction and repeat the same procedure 3 more times.rnFilter on 0.2um filter.rnImmediately use or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.