Direct ELISA analysis of TNF alpha was performed by coating wells of a 96-well plate with 100µl per well of human recombinant protein TNF alpha (Product # RTNFAI) diluted in carbonate/bicarbonate buffer (Product # 28382) at a concentration of 1µg/ml overnight at 4C. Wells of the plate were washed, blocked with starting blocking buffer (Product # 37538), and incubated with 100µl per well of a rabbit anti-TNF alpha monoclonal antibody (Product # M302) at a dilution of 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200 for 90 minutes at 37C. The plate was washed, then incubated with 100µl per well of a HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 65-6120) at a dilution of 1:2000 for 90 minutes at 37C. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 5-10 minutes at room temperature in the dark. The reaction was stopped with Stop solution (Product #N600), and absorbances were read on a spectrophotometer at 450-550nm.
|Tested species reactivity||Human|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant protein corresponding to amino acids 77–233 of human TNF-alpha|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
P300A was successfully used to detect human TNF alpha in ELISA application.
This antibody is predicted to react with mouse, rat and non-human primate based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Tumor necrosis factor-alpha is a pleiotropic inflammatory cytokine that stimulates the acute phase reaction. The primary role of TNF is in the regulation of immune cells. Other functions of TNF-alpha include its role in the immune response to bacterial, viral, parasitic and certain fungal infections, as well as its role in the necrosis of specific tumors. TNF-alpha is a trimeric protein encoded within the major histocompatibility complex.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Mouse||Not Cited||Retinol dehydrogenase 13 protects the mouse retina from acute light damage.||Wang H,Cui X,Gu Q,Chen Y,Zhou J,Kuang Y,Wang Z,Xu X||Molecular vision (18:1021)||2012|