Recombinant human TNF alpha protein (Active)

• Flow Cyt

Lab

Flow Cytometry - Recombinant human TNF alpha protein (Active) (AB259410)

Flow cytometry overlay histogram showing wild-type A549 (green line) and VCAM1 knockout A549 cells (red line, ab273758), treated with 10 ng/ml TNF-alpha for 16 h (left) and untreated (right), stained with ab103173. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab103173) (1x10⁶ in 100μl at 0.2μg/ml) for 30 min at 4°C.

Isotype control antibody mouse IgG1κ Allophycocyanin was used at the same concentration and conditions as the primary antibody (wild-type A549 - black line VCAM knockout A549 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 40 mW Red laser (638nm) and 660/10 bandpass filter.

• sELISA

Unknown

Sandwich ELISA - Recombinant human TNF alpha protein (Active) (AB259410)

Sandwich ELISA - Recombinant human TNF alpha protein standard curve.

Background subtracted standard curve using Human TNF alpha Antibody Pair - BSA and Azide free (ab241791) and Recombinant human TNF alpha protein (Active) (ab259410) in sandwich ELISA. The ELISA was performed using the components of the corresponding SimpleStep® kit, which uses the same antibody pair with a different formulation and format.

• Mass Spec

Supplier Data

Mass Spectrometry - Recombinant human TNF alpha protein (Active) (AB259410)

M + 0.2 Da (calc. mass 17409.8 Da)

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).

• FuncS

Supplier Data

Functional Studies - Recombinant human TNF alpha protein (Active) (AB259410)

Fully biologically active when compared to standard. The ED₅₀ as determined by the dose-dependant Killing/apoptosis of L-929 cells is 0.71 ng/mL corresponding to a Specific Activity of 1.41 x 10⁶ IU/mg.

• sELISA

Lab

Sandwich ELISA - Recombinant human TNF alpha protein (Active) (AB259410)

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).

• SDS-PAGE

Supplier Data

SDS-PAGE - Recombinant human TNF alpha protein (Active) (AB259410)

SDS-PAGE analysis of ab259410.

• HPLC

Supplier Data

HPLC - Recombinant human TNF alpha protein (Active) (AB259410)

Purity : 100%

The spectrum was recorded using a 1260 Infinity II HPLC system with DAD and a MabPac RP column (3.0x100 mm, 4 μm). 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.