Rat Adiponectin ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents and samples to room temperature (18–25 °C) before use.	-Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. -Read at 450 nm immediately after adding STOP solution.

Upstream tips
-Bring all reagents and samples to room temperature (18–25 °C) before use.
Protocol tips
-Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. -Read at 450 nm immediately after adding STOP solution.

Publication protocol

A rat adiponectin ELISA kit (Sigma-Aldrich, St. Louis, MO, USA) was used to measure adiponectin concentration in blood plasma according to the manufacturer’s protocol. Briefly, 1× wash buffer was prepared by diluting the 20× wash buffer with deionized water and the 1× ELISA buffer was also prepared by diluting the 5× ELISA buffer with deionized water. The rat adiponectin protein standard was reconstituted with 1 mL of deionized water and serial dilutions of the standard were prepared. Then 1× biotinylated rat adiponectin detection antibody solution was prepared by diluting the detection antibody 1:1000 in 1× ELISA buffer and 1× HRP-Streptavidin solution was prepared by diluting HRP solution 1:100 in 1× ELISA buffer.

The protein standard and samples were added into appropriate wells (100 µL to each well) and the plate was incubated at room temperature for 2.5 h with gentle shaking. The solution was discarded and the wells were washed 4 times with 300 µL 1× wash solution. Then 100 µL of 1× prepared biotinylated detection antibody was added to each well and the plate was incubated for 1 h at room temperature with gentle shaking. The solution was discarded and the wells were washed 4 times with 300 µL 1× wash solution again. Then 100 µL of the prepared HRP-Streptavidin solution was added to each well and the plate was incubated for 45 min at room temperature with gentle shaking. The solution was discarded and the wells were washed 4 times with 300 µL 1× wash solution for the last time. Then 100 µL of ELISA colorimetric TMB reagent was added to each well and the plate was incubated for 30 min at room temperature in a dark room with gentle shaking. Lastly, 50 µL of stop solution was added to each well and absorbance was measured at 450 nm immediately with a spectrophotometer. The protein standard curve was generated and the unknown concentration of the samples was determined.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Rat Adiponectin ELISA Kit below.

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