Human HIF-1 alpha Recombinant Rabbit Monoclonal Antibody [PSH26-18] - BSA and Azide free (Capture/Detector)

Sandwich ELISA analysis of Human HIF-1 alpha matched pair antibodies

Capture: HA724603, Human HIF-1 alpha Rabbit mAb [PSH26-18]
Detector: HA724604, Human HIF-1 alpha Rabbit mAb [PSH26-19]

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA724603) diluted in carbonate/bicarbonate buffer, at a concentration of 2 μg/mL overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1% BSA blocking buffer, and incubated with serial diluted Recombinant Human HIF-1 alpha protein (HA211604) starting from 5,000 pg/mL to 0 pg/mL and detect antibody (HA724604, Biotin, 0.2 μg/mL) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Sandwich ELISA analysis of Human HIF-1 alpha matched pair antibodies

Capture: HA724605, Human HIF-1 alpha Rabbit mAb [PSH26-20]
Detector: HA724603, Human HIF-1 alpha Rabbit mAb [PSH26-18]

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA724605) diluted in carbonate/bicarbonate buffer, at a concentration of 2 μg/mL overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1% BSA blocking buffer, and incubated with serial diluted Recombinant Human HIF-1 alpha protein (HA211604) starting from 5,000 pg/mL to 0 pg/mL and detect antibody (HA724603, Biotin, 0.2 μg/mL) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Interpolated concentrations of native HIF-1 alpha in MCF7 cell extract samples based on a 1,000 µg/mL extract load.

Capture: HA724603, Human HIF-1 alpha Rabbit mAb [PSH26-18]
Detector: HA724604, Human HIF-1 alpha Rabbit mAb [PSH26-19]

MCF7 cells were treated with or without CoCl2 and incubated for 8 hours. The concentrations of HIF-1 alpha were measured in duplicates, interpolated from the HIF-1 alpha standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean HIF-1 alpha concentration was determined to be 177 pg/mL in untreated MCF7 cell extract and 1,977 pg/mL in CoCl2 treated MCF7 cell extract.

Interpolated concentrations of native HIF-1 alpha in MCF7 cell extract samples based on a 1,000 µg/mL extract load.

Capture: HA724605, Human HIF-1 alpha Rabbit mAb [PSH26-20]
Detector: HA724603, Human HIF-1 alpha Rabbit mAb [PSH26-18]

MCF7 cells were treated with or without CoCl2 and incubated for 8 hours. The concentrations of HIF-1 alpha were measured in duplicates, interpolated from the HIF-1 alpha standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean HIF-1 alpha concentration was determined to be 165 pg/mL in untreated MCF7 cell extract and 2,160 pg/mL in CoCl2 treated MCF7 cell extract.

Interpolated concentrations of spiked HIF-1 alpha in human cell culture media samples.

Capture: HA724603, Human HIF-1 alpha Rabbit mAb [PSH26-18]
Detector: HA724604, Human HIF-1 alpha Rabbit mAb [PSH26-19]

The concentrations of HIF-1 alpha were measured in duplicates, interpolated from the HIF-1 alpha standard curves and corrected for sample dilution. Diluted samples are as follows: 50% cell culture media with FBS. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).

Interpolated concentrations of spiked HIF-1 alpha in human cell culture media samples.

Capture: HA724605, Human HIF-1 alpha Rabbit mAb [PSH26-20]
Detector: HA724603, Human HIF-1 alpha Rabbit mAb [PSH26-18]

The concentrations of HIF-1 alpha were measured in duplicates, interpolated from the HIF-1 alpha standard curves and corrected for sample dilution. Diluted samples are as follows: 50% cell culture media with FBS. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).