GM-CSF Human ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-All samples and reagents must be at room temperature (20-25°C) before use in the ELISA. -Do not use water baths to thaw samples. Thaw at room temperature.	-When preparing standard curve and sample dilution in culture medium, use the same medium used to culture the cells. -Once reagents have been added to the plate, take care NOT to let plate DRY at any time during the assay. -If the TMB substrate solution is blue before use, DO NOT USE IT. -Prepare Streptavidin-HRP Solution immediately before use.

Upstream tips
-All samples and reagents must be at room temperature (20-25°C) before use in the ELISA. -Do not use water baths to thaw samples. Thaw at room temperature.
Protocol tips
-When preparing standard curve and sample dilution in culture medium, use the same medium used to culture the cells. -Once reagents have been added to the plate, take care NOT to let plate DRY at any time during the assay. -If the TMB substrate solution is blue before use, DO NOT USE IT. -Prepare Streptavidin-HRP Solution immediately before use.

Publication protocol

Previously described procedures were used for the ELISAs with some modifications (32, 33). EIA/RIA plates (Corning Incorporated, Corning, NY) were coated with 100 μL of purified recombinant protein at a concentration of 5 μg/mL in coating buffer (0.05% sodium azide containing PBS) overnight at 4°C. Angiopoietin-1 and angiopoietin-2 were from R&D, tissue-type plasminogen activator was from Abnova, and recombinant ML-IAP and NY-ESO-1 were prepared in house. The plates were washed with PBST (0.05% Tween-20 containing PBS) and blocked for two hours at room temperature with 200 μL/well blocking solution (PBST, 2% nonfat milk, 0.05% sodium azide). After the plates were again washed, longitudinal sera samples were added at a final dilution of 1:500 in blocking solution (100 μL/well) and incubated at 4°C overnight. After several further washes, the plates were incubated with 100 μL/well of a 1:2000 diluted alkaline phosphatase–conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for one hour at room temperature. Finally, the plates were washed again, incubated with 100 μL/well of the PNPP substrate (Sigma, St. Louis, MO) for 25 minutes at room temperature, and then the OD (405 nm) was determined (Spectramax 190 Microplate Reader; Molecular Devices, Sunnyvale, CA).

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Manufacturer protocol

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