Monoclonal mouse anti human TNF-alpha

///Monoclonal mouse anti human TNF-alpha

Monoclonal mouse anti human TNF-alpha

$99.00$149.00


accession P01375


Applications

ELISA:This antibody can be used at 4 μg/mL with the appropriate secondary reagents to detect TNF alpha. The detection limit for recombinant human TNF alpha is approximately0.1 ng/well.

Source This antibody was produced from a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse immunized with recombinant E. coli-derived Human TNFa.
Species reactivity Human
Purification The IgG fraction of the tissue culture supernatant was purified by Protein G affinity chromatography.
Presentation Lyophilized from sterile filtered solution at a concentration of 1mg/ml in PBS pH 7.4.
Reconstitution A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than 0.1 mg/mL. This solution can then be diluted into other buffers
Storage The lyophilized antibody is stable for at least 1 year from date of receipt at -20° C. Upon reconstitution, this antibody can be stored in working aliquots at 2° - 8° C for one month, or at -20° C for six months without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This antibody product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Interactor O88351
Interactor O88522
Interactor O89110
Interactor P01374 TNFB_HUMAN
Interactor P01730 CD4_HUMAN
Interactor P25118 TNR1A_MOUSE
Interactor P39429
Interactor P62991
Interactor Q12933
Interactor Q15628
Interactor Q60680
Interactor Q60769
Interactor Q60855
Interactor Q62073
Interactor Q924T7
Interactor Q9QZL0
Interactor P19438 TNR1A_HUMAN
Interactor P20333 TNR1B_HUMAN
Interactor P25118 TNR1A_MOUSE
Interactor P36941 TNR3_HUMAN
Interactor P70191
Interactor Q13546
Interactor Q3U0V2
Interactor Q80TQ2
Interactor Q9DHW0
Interactor Q9QZL0
Molecular function Cytokine

Methods

Immunohistochemistry

  • The following anti-human primary antibodies and dilutions were used: rabbit anti-C3 polyclonal antibody (pAb) at a dilution of 1:100 , mouse anti-C4 monoclonal antibody (mAb) at 1:100 (clone IF6; Novocastra, Newcastle-upon-Tyne, UK), mouse anti-C8 mAb at 1:100 (clone 144B), mouse anti-C20 mAb at 1:400 (clone ), mouse anti-C68 mAb at 1:200 (clone Kp-1; , , , ), mouse anti-c-kit mAb at 1:100 (clone 1042), mouse anti-IL-1β mAb at 1:500 (clone 8516& , , , ), mouse anti-IL-6 mAb at 1:500 (clone 1936& ), mouse anti-TNF-α mAb at 1:100 (clone 28401& ), mouse anti-ANKL mAb at 1:100 (clone 70525& ), mouse anti-ANK mAb at 1:400 (clone 80707& ), and goat anti-OPG pAb at 1:20 .

iNKT cell activation assays

  • Activation of iNKT cells after coculture with DCs from 81A, 81A-eGFP, or mock-infected cultures was assessed by flow cytometry and microscopy.
  • For the flow cytometry assay, DCs were preincubated with different concentrations of α-GalCer (0.1–100 ng/mL) overnight at 37°C before adding iNKT cells at a ratio of 1:2.
  • After 6 h of coculture in the presence of brefeldin A (2 mg/mL ), cells were surface-stained with anti-CD3 and anti-CD11c mAb to discriminate between DCs and iNKT cells.
  • Following permeabilization, cells were stained with anti-IFN-γ mAb and analyzed on a .
  • To assess iNKT cell activation by microcopy, iNKT cells were coincubated with DCs preloaded with α-GalCer (100 ng/mL) at a 1:2 ratio in the presence of brefeldin A.
  • Minimal coincubation times for complex formation and cytokine detection were 30 min for TNF-α and 2 h for IFN-γ.
  • After fixation with 4% paraformaldehyde, cell complexes were cytospun onto glass…
  • Activation of iNKT cells after coculture with DCs from 81A, 81A-eGFP, or mock-infected cultures was assessed by flow cytometry and microscopy.
  • For the flow cytometry assay, DCs were preincubated with different concentrations of α-GalCer (0.1–100 ng/mL) overnight at 37°C before adding iNKT cells at a ratio of 1:2.
  • After 6 h of coculture in the presence of brefeldin A (2 mg/mL ), cells were surface-stained with anti-CD3 and anti-CD11c mAb to discriminate between DCs and iNKT cells.
  • Following permeabilization, cells were stained with anti-IFN-γ mAb and analyzed on a .
  • To assess iNKT cell activation by microcopy, iNKT cells were coincubated with DCs preloaded with α-GalCer (100 ng/mL) at a 1:2 ratio in the presence of brefeldin A.
  • Minimal coincubation times for complex formation and cytokine detection were 30 min for TNF-α and 2 h for IFN-γ.
  • After fixation with 4% paraformaldehyde, cell complexes were cytospun onto glass microscope slides using a Cytospin 4 cytoentrifuge (800 rpm, 5 min , , ) and permeabilized with 0.1% saponin .
  • Cells were stained with anti-IFN-γ mAb (25,718; 1:50& ) or anti-TNF-α mAb (28,401; 1:50& ), blocked with 1% normal goat serum (ako, , enmark), and finally, incubated with goat anti-mouse Alexa-594 secondary antibody (1:500 , , O, ) and API .
  • All incubations with primary and secondary antibodies and serum were at room temperature for 30 min.
  • Images were obtained on a Nikon A1R confocal system with a 60×/1.49 oil objective using NIS-Elements AR software (3.2).
  • IFN-γ production by iNKT cells was analyzed by visual inspection of randomly taken images of DC-iNKT cell complexes.
  • To avoid operator bias, image acquisition and analysis were performed independently by different investigators.

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