Rat beta NGF ELISA Kit (ab193736)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Equilibrate all reagents to room temperature (18-25°C) prior to use.	-Avoid foaming or bubbles when mixing or reconstituting components. -Ensure plates are properly sealed or covered during incubation steps. -Complete removal of all solutions and buffers during wash steps is necessary to minimize background.	-Unused well strips should be returned to the plate packet and stored at 4°C.

Upstream tips
-Equilibrate all reagents to room temperature (18-25°C) prior to use.
Protocol tips
-Avoid foaming or bubbles when mixing or reconstituting components. -Ensure plates are properly sealed or covered during incubation steps. -Complete removal of all solutions and buffers during wash steps is necessary to minimize background.
Downstream tips
-Unused well strips should be returned to the plate packet and stored at 4°C.

Publication protocol

The rats were intraperitoneally injected with 2% pentobarbital sodium (WS20051129, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and 4 ∼ 5 ml of blood was extracted from the abdominal aorta, placed at room temperature for 30 min, and centrifuged at 3500 r/min for 15 min to separate the serum. The assay was performed strictly in accordance with the instructions of the tumor necrosis factor-α (TNF-α) ELISA kit (ab46070), interleukin-1β (IL-1β) ELISA kit (ab100768), IL-6 ELISA kit (ab100772), IL-10 ELISA kit (ab100764) and nerve growth factor (NGF) ELISA kit (ab193736) purchased from Abcam Inc. (Cambridge, MA, USA) and the neurite outgrowth inhibitor -A (Nogo-A) ELISA kit (CSB-EQ027219RA, Shanghai Gaochuang Chemical Co., Ltd., Shanghai, China). The ELISA kit was balanced at room temperature for 20 min. After the standard substances were dissolved, 100 µl was added into the reaction plate to draw the standard curve. A total of 100 µl of sample was added to the reaction well, followed by incubation at 37°C for 90 min and 5 washes at intervals of 30 s. Then, 100 µl of fresh biotinylated antibody working fluid was added, followed by incubation at 37°C for 1 h and 5 washes at intervals of 30 s. After the washes, 100 µl of fresh enzyme-bound reactant working fluid was added away from light, followed by another incubation at 37°C for 30 min and 5 washes. Then, stop buffer was swiftly added to terminate the reaction. The optical density (OD) value of each well was determined at the wavelength of 450 nm using a microplate reader (ELx800, Bio-TEK Instruments Inc., VT, USA) within 3 min. The standard curve was drawn according to the OD value, and the contents of inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10), NGF and Nogo-A were evaluated. The experiment was repeated 3 times.



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Manufacturer protocol

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