Tested samples should be clear and transparent and be centrifuged to remove suspended solids before experiment.
Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 15 min at 1000×g at 2~8℃. Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 min at 1000×g at 2~8℃ within 30 min of collection. Collect the supernatant and carry out the assay immediately. Hemolysis samples are not suitable for ELISA assay!
Cell lysates: For adherent cells, gently wash the cells with moderate amount of pre-cooled PBS and dissociate the cells by trypsin. Collect the cell suspension into the centrifugal tube and centrifuge for 5 min at 1000×g. Discard the medium and wash the cells for 3 times with pre-cooled PBS. For each 1×106 cells, add 150-250 μL of pre-cooled PBS to keep the cells resuspended. Repeat the freeze-thaw process for several times until the cells are lysed fully. Centrifuge for 10min at 1500×g at 4℃. Remove the cell fragments, collect the supernatant and store at -20℃ or -80℃. Avoid repeated freeze-thaw cycles.
Tissue homogenates: It is recommended to get detailed references from other literatures before detecting different tissue types. For general information, hemolysis blood may affect the result, so the tissues should be minced to small pieces and rinsed in ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (the volume depends on the weight of the tissue) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5 min at 50000×g to get the supernatant.
Cell culture supernatant or other biological fluids: Centrifuge samples for 20 min at 1000×g at 2~ 8℃. Collect the supernatant and carry out the assay immediately.
1. Samples should be assayed within 7 days when stored at 4℃, otherwise samples must be divided and stored at -20℃ (≤1 month) or -80℃ (≤3 months). Avoid repeated freeze-thaw cycles.
2. Please predict the concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
3. If the sample type is not included in the manual, a preliminary experiment is suggested to verify the validity.
4. If lysis buffer is used to prepare tissue homogenate or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance.
5. Some recombinant protein may cannot be detected due to the mismatching with coated antibody or detection antibody.