Rat/Mouse Cytochrome c Quantikine ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.	-When mixing or reconstituting protein solutions, always avoid foaming. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution.

Upstream tips
-Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Protocol tips
-When mixing or reconstituting protein solutions, always avoid foaming. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution.

Publication protocol

Cells were scraped, washed in 1XPBS, resuspended in 1× extraction buffer A containing 10 mM HEPES, pH 7.5, 200 mM mannitol, 70 mM sucrose and 1 mM EGTA (Sigma) and 2 mg per ml bovine serum albumin (Sigma) and kept on ice for 1 h. Cells were lysed by a B-type Dounce homogenizer, and homogenates were centrifuged at 4 °C in the subsequent steps to remove nuclei, debris, and mitochondria. To obtain the cytosolic fraction, supernatant was centrifuged at 15,000 g at 4 °C. The cytochrome c concentration in the cytosolic fraction was quantified by solid phase ELISA kit (Quantikine Rat/Mouse, R & D systems) according to the manufacturer’s protocol.

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Manufacturer protocol

Download the product protocol from R&D Systems for Rat/Mouse Cytochrome c Quantikine ELISA Kit below.

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