Recombinant Human Interferon gamma protein (Active) (ab259377)

Wild-type A549 control cells or IP-10 knockout A549 cells (ab266969), grown to 40% confluency, were stimulated with Recombinant Human Interferon gamma protein (ab259377) at 100 ng/ml and Recombinant human TNF alpha protein (ab259410) at 10 ng/ml or vehicle control for 16 or 32 hours.

THP-1 cells, grown to 40% confluency, were stimulated with Recombinant Human Interferon gamma protein (ab259377) at 200 ng/ml and LPS at 50 ng/mL or vehicle control for 24 hours.

The concentrations of IP-10 (CXCL10) in cell culture supernatants were measured in duplicate and interpolated from the IP-10 standard curves using Human IP-10 ELISA Kit (ab173194). IP-10 from vehicle control samples were measured in undiluted supernatants and the treated samples were diluted 200 times. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).

Fully biologically active determined by the dose dependent reduction in cytopathic effect of viral infection in A549 cells. ED₅₀ for this effect is 0.30 ng/ml corresponding to a specific activity of 3.33 x 10⁶ units/mg.

SDS-PAGE analysis of ab259377.

Lanes 1 - 6: Merged signal (red and green). Green - ab214668 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab214668 was shown to react with IP10 in wild-type A549 cells in western blot with loss of signal observed in IP10 knockout cell line ab266971 (knockout cell lysate ab256888). Wild-type and IP10 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214668 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1- 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab213524 was shown to react with PD-L1 in wild-type A549 treated with 100 ng/ml IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell lines ab267054 ( treated and untreated knockout cell lysates ab256831) were used. Wild-type A549 treated with 100 ng/ml IFN gamma for 48 h and CD274 knockout A549 treated with 100 ng/ml IFN gamma for 48 h cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 4: Merged signal (red and green). Green - ab137018 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab137018 was shown to react with IP10 in A549 wild-type cells in western blot with loss of signal observed in IP10 knockout cell line ab266969 (IP10 knockout cell lysate ab256886). A549 wild-type and IP10 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab137018 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Purity: 100%

The spectrum was recorded using a 1260 Infinity II HPLC system with DAD (Agilent Technologies) and a MabPac RP column (3.0x100 mm, 4 µm, Thermo Scientific). 5 µL of purified protein was injected and the gradient run from 80 % water:TFA (99.9:0.1 v/v) and 20 % acetonitrile:water:TFA (90:9.9:0.1 v/v/v) to 20 % water:TFA (99.9:0.1 v/v) and 80 % acetonitrile:water:TFA (90:9.9:0.1 v/v/v) within 3 minutes followed by an isocratic step for another 3 min. Flow rate was 0.5 mL/min and the column compartment temperature was 50 °C.

Mass Spectrometry analysis of ab259377 (Agilent Technologies). The mass spectrum shows the full length protein and two C-terminal truncated versions (-5 aa and -16 aa). Heterogeneity of C-terminus has been reported in literature (PAN, Yu‐Ching E., et al. "Structural characterization of human interferon γ Heterogeneity of the carboxyl terminus." European journal of biochemistry 166.1 (1987): 145-149. ).

M + 1.4 Da (calc, mass 16833.6)

The spectrum was recorded with a 6545XT AdvanceBio LC/Q-TOF (Agilent Technologies) and a MabPac RP column (42.1x50 mm, 4 µm, Thermo Scientific). 5 µL of purified protein was injected and the gradient run from 85 % water:FA (99.9:0.1 v/v) and 15 % acetonitrile:FA (90:9.9:0.1 v/v/v) to 55 % water:FA (99.9:0.1 v/v) and 45 % acetonitrile:FA (90:9.9:0.1 v/v/v) within 3 minutes followed by an isocratic step for another 2.5 min. Flow rate was 0.4 mL/min and the column compartment temperature was 60 °C. Data was analysed and deconvoluted using the Bioconfirm software (Agilent Technologies).