Mouse GM-CSF DuoSet ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.	-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Upstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.
Protocol tips
-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Publication protocol

Mouse serum GM-CSF levels were measured with an inhouse ELISA method (DY415-05; R&D Systems, Minneapolis,
MN, USA). In brief, the GM-CSF capture antibody was coated
onto NUNC Maxisorp plates (Sigma-Aldrich) at 50 ng/well
maintained at 4°C overnight before washing with 200 ml PBSTween (PBST; containing 1% bovine serum albumin to block
the plate) for 2 h at room temperature. After washing twice in
200 ml PBST, mice serum samples and the GM-CSF standard were loaded to the plate and incubated at room temperature for 1 h. After 2 washes in 200 ml PBST, 100 ml detection antibody with 1:5000 dilutions in PBST with 1% bovine serum albumin (BSA) was added for an additional 30-min incubation
at room temperature. Finally, streptavidin-HRP with 1:10,000 dilution in PBST containing 1% BSA was added and mixed for 10 min at room temperature. Finally, after 3 washes in 200 ml PBST, 100 ml 3,39,5,59-tetramethylbenzidine was added and
reacted for 10 min at room temperature before the reaction was stopped with 50 ml2NH2SO4 and the results read at an optic density of 450 nm.

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Manufacturer protocol

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