|Applications||ELISA, IHC, WB|
|Product Name||Anti-TNF alpha Antibody|
|Storage & Handling||At -20°C for one year. After reconstitution, at 4°C for one month. It can also be aliquotted and stored frozen at -20°C for a longer time.Avoid repeated freezing and thawing.|
|Description||Rabbit IgG polyclonal antibody for Tumor necrosis factor(TNF) detection. Tested with WB, IHC-P, ELISA in Mouse.|
|Cite This Product||Anti-TNF alpha Antibody (Boster Biological Technology, Pleasanton CA, USA, Catalog # RP1000)|
|Immunogen||E. coli-derived mouse TNF alpha recombinant protein(Position: L80-L235).|
Assay Dilutions Overview
ELISA , 0.1-0.5μg/ml, Mouse, -
Western blot, 0.1-0.5μg/ml, Mouse
Boster's Secondary Antibodies And IHC, WB Kits
The following reagents are used to generate the images below.Boster recommends Enhanced Chemiluminescent Kit with anti-Rabbit IgG (EK1002) for Western blot, and HRP Conjugated anti-Rabbit IgG Super Vision Assay Kit (SV0002-1) for IHC(P).
Images And Assay Conditions
Figure. Western blot analysis of TNF alpha using anti- TNF alpha antibody (RP1000).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V(Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane: Recombinant Mouse TNFα Protein 0.5ng,
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti- TNF alpha antigen affinity purified polyclonal antibody (Catalog # RP1000) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for TNF alpha at approximately 17KD. The expected band size for TNF alpha is at 17KD.
Protein Target Info (Source: Uniprot.org)
|Protein Name||Tumor necrosis factor|
|Alternative Names||Tumor necrosis factor;Cachectin;TNF-alpha;Tumor necrosis factor ligand superfamily member 2;TNF-a;Tumor necrosis factor, membrane form;N-terminal fragment;NTF;Intracellular domain 1;ICD1;Intracellular domain 2;ICD2;C-domain 1;C-domain 2;Tumor necrosis factor, soluble form;Tnf;Tnfa, Tnfsf2;|
|Subcellular Localization||Cell membrane; Single-pass type II membrane protein.|
|Molecular Weight||25896 MW|
*if product is indicated to react with multiple species, protein info is based on the human gene.
|Protein Function||Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation.|
|Background||TNF alpha(Tumor Necrosis Factor alpha) gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor(TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Knockout studies in mice also suggested the neuroprotective function of this cytokine.|
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1. Post-translational modification:phosphorylation, methylation, glycosylation etc. These modifications prevent SDS molecules from binding to the target protein and thus make the band size appear larger than expected
2. Post-translational cleavage: this can cause smaller bands and or multiple bands
3. Alternative splicing: the same gene can have alternative splicing patterns generating different size proteins, all with reactivities to the antibody.
4. Amino Acid R chain charge: SDS binds to positive charges. The different size and charge of the Amino Acid side chains can affect the amount of SDS binding and thus affect the observed band size.
5. Multimers: Multimers are usually broken up in reducing conditions. However if the interactions between the multimers are strong, the band may appear higher.,