Human HSP70/HSPA1A 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

Detection of Hsp70 or CD63 in total exosomes lysates was performed using specific ELISA kits from R&D Systems (DY1663–2) and BioSource (MBS763944), respectively. Plates were developed using a peroxidase substrate system (R&D Systems), and then read with the Victor3 multilabel plate reader (Model # 1420–033, Perkin Elmer, Santa Clara, CA) capable of measuring absorbance in 96-well plates using dual wavelength of 450–540 nm. Results were expressed as picograms per milliliter (pg/mL) and referred to a standard curve obtained by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and drawing a best-fit curve through the points on the graph.

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Manufacturer protocol

Download the product protocol from R&D Systems for Human HSP70/HSPA1A DuoSet ELISA below.

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