Egf human elisa kit

HP-B hydrogel was pre-mixed with 50 ng ml^–1 EGF. 1.5 mL of this pre-mixed hydrogel was added to a 12-well plate (n = 3) and allowed to gelate for about 4 to 5 hours at room temperature. Following this, 1.5 mL of serum-free, EGF-free growth media was added on top of the hydrogel in each of the wells and incubated at 37 °C. The supernatant was collected, and the wells were replenished with 1.5 ml of fresh serum-free, EGF-free media every 24 hours for a total of 72 hours. The collected supernatants were analyzed for total EGF content, using EGF Human ELISA kit (Invitrogen, KHG0061). Previous studies have validated a normalization factor to account for the degradation of the protein in heparin-based hydrogel, that was used to post-process our data from ELISA.36 (link)

Serum and urinary EGF were measured using an EGF human Elisa kit (Invitrogen, California, USA), according to the manufacturer’s guidelines. The detection limit of this assay was 3.9 pg/ml. A preliminary experiment (n = 10) was performed to test the intra- and inter-variability of the EGF human Elisa kit, showing a mean intra-assay coefficient of variance of 9.82% and an inter-assay coefficient of variation of 9.81%.
Urinary EGF was corrected for urinary creatinine, in order to correct for differences in urine concentration, analogous to previous studies [3 (link), 5 (link)]. The correction for urinary creatinine is expressed as “uCr”.

Urinary EGF was measured using an EGF human Elisa kit^® (Invitrogen, Waltham, MA, USA), according to the manufacturer’s guidelines. The detection limit of this assay was 3.9 pg/mL. A preliminary experiment (n = 10) was performed to test the intra- and inter-variability of the EGF human Elisa kit^®, showing a mean intra-assay coefficient of variance of 9.82% and an inter-assay coefficient of variation of 9.81%.

Cells grown in culture medium to 70% confluency were starved in serum-free medium for 16 h and then transferred to 4 °C prior to the addition of 50 ng/ml biotinylated EGF (Invitrogen). Cells were washed 3 times with ice-cold PBS to remove unbound biotinylated EGF and then incubated at 37 °C for various time periods to allow EGF internalization. biotinylated EGF not yet internalized was blocked with free avidin (0.05 mg/ml, Invitrogen) on ice and washed 3 times with ice-cold PBS to remove the unbound avidin. The internalized EGF was analyzed by Human EGF ELISA Kit (Invitrogen) according to manufacturer’s instruction, followed by absorbance measurement at 450 nm with Infinite M1000 pro (Tecan).