Immunoassays for Aβ1-40 (ThermoFisher, KHB3481), Aβ1-42 (ThermoFisher KHB3441), aggregated Aβ (ThermoFisher, KHB3491), and sAβPP-α (MyBioSource, MBS9358454) in conditioned media was measured via ELISA according to manufacturer’s instruction. Total 8-isoprostanes were measured via ELISA (516351, Cayman Chemical) in cell lysates following purification using the 8-isoprostane affinity sorbent (401113, Cayman Chemical) suspended in Eicosanoid Column Affinity buffer (400220, Cayman Chemical). Protein carbonylation was assessed by the method of Carty et al. (2000).
Eicosanoid column affinity buffer
Manufactured by Cayman Chemical
The Eicosanoid Column Affinity buffer is a laboratory product designed to facilitate the purification and separation of eicosanoid compounds. It serves as a crucial component in the analysis and study of these important biological molecules.
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3 protocols using eicosanoid column affinity buffer
1
Quantifying Alzheimer's Biomarkers
2
Plasma 8-isoprostane Quantification by ELISA
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Total 8-isoprostanes were measured using an ELISA kit to assess lipid peroxidation in plasma (8-isoprostane ELISA kit, Cayman Chemical). Prior to this assay the plasma samples were purified following the manufacturer's instructions.
Briefly, plasma (100 µL) was added to the 8-isoprostane affinity sorbent (401113, Cayman Chemical) and incubated for 60 minutes with gentle mixing and then centrifuged at 1500xg for 30 seconds to sediment the sorbent. The supernatant was removed and discarded. Eicosanoid Column Affinity buffer (100μL, 400220, Cayman Chemical) was added and placed in the centrifuge at 1500xg for 30 seconds, the supernatant was then removed. This step was repeated by adding ultrapure water (100µL) before centrifugation and discarding the supernatant. Elution solution (100µL, 95% ethanol) was then added to the sediment and evaporated to dryness under nitrogen. Samples were suspended in the ELISA buffer (100µL). Next, standards were prepared from the assay stock solution to create an 8-point standard curve ranging from 0.8 to 500pg/ml, a blank well was used as the 0pg.ml standard.
Standard or sample (50µL) was added to the appropriate well with of 8-isoprostane tracer (50µL) and antiserum (50µL). This was incubated for 18hours at 4 O C. Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
Briefly, plasma (100 µL) was added to the 8-isoprostane affinity sorbent (401113, Cayman Chemical) and incubated for 60 minutes with gentle mixing and then centrifuged at 1500xg for 30 seconds to sediment the sorbent. The supernatant was removed and discarded. Eicosanoid Column Affinity buffer (100μL, 400220, Cayman Chemical) was added and placed in the centrifuge at 1500xg for 30 seconds, the supernatant was then removed. This step was repeated by adding ultrapure water (100µL) before centrifugation and discarding the supernatant. Elution solution (100µL, 95% ethanol) was then added to the sediment and evaporated to dryness under nitrogen. Samples were suspended in the ELISA buffer (100µL). Next, standards were prepared from the assay stock solution to create an 8-point standard curve ranging from 0.8 to 500pg/ml, a blank well was used as the 0pg.ml standard.
Standard or sample (50µL) was added to the appropriate well with of 8-isoprostane tracer (50µL) and antiserum (50µL). This was incubated for 18hours at 4 O C. Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
3
Quantification of 8-Isoprostane: Lipid Peroxidation Marker
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Total 8-isoprostanes were measured using a commercially available ELISA kit to assess lipid peroxidation (8-isoprostane ELISA kit, Cayman Chemical). Prior to this assay the cell lysate samples were purified following the manufacturer's instructions. Briefly, cell lysate was added to the 8-isoprostane affinity sorbent (401113, Cayman Chemical) and incubated for 60 minutes with gentle mixing. Samples were then centrifuged at 1500xg for 30 seconds to sediment the sorbent, the resulting supernatant was removed and discarded, this was repeated 2 times following the addition Eicosanoid Column Affinity buffer (100μL, 400220, Cayman Chemical) and ultrapure water (100μL). Elution solution (95% ethanol) was then added to the sediment and evaporated to dryness under nitrogen. Samples were suspended in the ELISA buffer. Standards were then prepared from the assay stock solution to create an 8-point standard curve ranging from 0.8 to 500pg/mL, ELISA buffer was used as the 0pg.mL standard. Standard or lysates (50µL) were added to the appropriate well with 8-isoprostane tracer (50μL) and antiserum (50μL) in triplicate, this was incubated for 18hours at 4°C.
Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
Ellman's reagent was added to each well (200μL) and left to incubate for 2 hours with gentle agitation. The plate was then read at 420nm.
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