Recombinant human TNF alpha protein (Active)

7 product images

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  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, Human VCAM1 knockout A549 cell line ab273758), treated with 10 ng/ml TNF-alpha for 16 h (left) and untreated (right), stained with APC Anti-VCAM1 antibody [STA] ab103173. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (APC Anti-VCAM1 antibody [STA] 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.

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  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 (Human/Monkey 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.

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  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.

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  Sandwich ELISA - Recombinant human TNF alpha protein (Active) (ab259410)

  Wild-type A549 control cells or IP-10 knockout A549 cells (Human CXCL10 (IP10) knockout A549 cell line ab266969), grown to 40% confluency, were stimulated with Recombinant Human Interferon gamma protein (Recombinant Human Interferon gamma protein (Active) 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 (Recombinant Human Interferon gamma protein (Active) 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 (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).

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  SDS-PAGE - Recombinant human TNF alpha protein (Active) (ab259410)

  SDS-PAGE analysis of ab259410.

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  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.

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  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).