Recombinant Anti-TNF alpha antibody [EPR22598-212] (ab255275)
![Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)](https://www.abcam.com/ps/products/255/ab255275/Images/ab255275-450513-anti-tnf-alpha-antibody-epr22598-212-western-blot-wildtype-thp1-brefeldin-a-treated-5-ugml-4-h-wildtype-thp1-lps-treate.jpg "Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR22598-212] to TNF alpha
- Suitable for: WB, IP, Flow Cyt (Intra)
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
-
Product name
Anti-TNF alpha antibody [EPR22598-212]
See all TNF alpha primary antibodies -
Description
Rabbit monoclonal [EPR22598-212] to TNF alpha -
Host species
Rabbit -
Tested applications
Suitable for: WB, IP, Flow Cyt (Intra)more details
Unsuitable for: ICC/IF or IHC-P -
Species reactivity
Reacts with: Mouse, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
-
Positive control
- WB: THP-1 (treated with 80nM TPA / 100 ng/ml lipopolysaccharide / 300ng/ml Brefeldin A) and RAW 264.7 (treated with 100 ng/ml lipopolysaccharide / 300 ng/ml Brefeldin A) whole cell lysates; Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A treated (5 µg/ml, 4 h) cell lysate. IP:THP-1 (treated with 80nM TPA / 100 ng/ml lipopolysaccharide / 300ng/ml Brefeldin A) whole cell lysate. Flow Cyt (intra): Human PBMC (treated with 50 ng/ml PMA/ 250 ng/ml ionomycon / 300 ng/ml Brefeldin A).
-
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
-
Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol (glycerin, glycerine), 0.05% BSA, PBS -
Concentration information loading... -
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR22598-212 -
Isotype
IgG -
Research areas
Associated products
-
Alternative Versions
-
Isotype control
-
KO cell lines
-
KO cell lysates
-
Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab255275 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
1/1000. Detects a band of approximately 25 kDa (predicted molecular weight: 25 kDa).
|
|
| IP |
1/30.
|
|
| Flow Cyt (Intra) |
1/700.
|
| Notes |
|---|
|
WB
1/1000. Detects a band of approximately 25 kDa (predicted molecular weight: 25 kDa). |
|
IP
1/30. |
|
Flow Cyt (Intra)
1/700. |
Target
-
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. -
Involvement in disease
Genetic variations in TNF are a cause of susceptibility psoriatic arthritis (PSORAS) [MIM:607507]. PSORAS is an inflammatory, seronegative arthritis associated with psoriasis. It is a heterogeneous disorder ranging from a mild, non-destructive disease to a severe, progressive, erosive arthropathy. Five types of psoriatic arthritis have been defined: asymmetrical oligoarthritis characterized by primary involvement of the small joints of the fingers or toes; asymmetrical arthritis which involves the joints of the extremities; symmetrical polyarthritis characterized by a rheumatoidlike pattern that can involve hands, wrists, ankles, and feet; arthritis mutilans, which is a rare but deforming and destructive condition; arthritis of the sacroiliac joints and spine (psoriatic spondylitis). -
Sequence similarities
Belongs to the tumor necrosis factor family. -
Post-translational
modificationsThe soluble form derives from the membrane form by proteolytic processing.
The membrane form, but not the soluble form, is phosphorylated on serine residues. Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.
O-glycosylated; glycans contain galactose, N-acetylgalactosamine and N-acetylneuraminic acid. -
Cellular localization
Secreted and Cell membrane. - Information by UniProt
-
Database links
- Entrez Gene: 7124 Human
- Entrez Gene: 21926 Mouse
- Omim: 191160 Human
- SwissProt: P01375 Human
- SwissProt: P06804 Mouse
- Unigene: 241570 Human
- Unigene: 1293 Mouse
-
Alternative names
- APC1 antibody
- APC1 protein antibody
- Cachectin antibody
see all
Images
-
Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution
Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 3 : TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Predicted band size: 25 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?Lanes 1 - 6: Merged signal (red and green). Green - ab255275 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab255275 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab255275 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
-
![Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [EPR22598-212] (ab255275)](https://www.abcam.com/ps/products/255/ab255275/Images/ab255275-347217-anti-tnf-antibody-epr22598-212-flowcytometry-pbmc-human.jpg)
Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [EPR22598-212] (ab255275)Intracellular flow cytometric analysis of2% paraformaldehyde-fixed, 0.1% Tween 20 permeabilized Human peripheral blood mononuclear cell (PBMC) treated with 50ng/ml PMA, 250ng/ml ionomycin and 300ng/ml Brefeldin A for 16h, labeling TNF alpha with ab255275 at 1/700 dilution (Right) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).
Goat Anti-Rabbit IgG Fc (Alexa Fluor® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.
Cells were surface stained with anti-CD3 conjugated to Alexa Fluor® 647. Then fixed with 2% PFA for 10min and intracellularly stained with rabbit IgG (Left) or ab236836 (Right).
-
![Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)](https://www.abcam.com/ps/products/255/ab255275/Images/ab255275-347220-anti-tnf-antibody-epr22598-212-western-blot-thp1-human.jpg)
Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution
Lane 1 : THP-1 (human monocytic leukemia cell line) (treated with 80nM 12-O-Tetradecanoylphorbol-13-acetate (TPA) overnight) whole cell lysate
Lane 2 : THP-1 (treated with 80nM TPA overnight, replaced the culture medium with 100 ng/ml lipopolysaccharides (LPS) for 6 hours with addition of 300ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 25 kDa
Observed band size: 25 kDa
Exposure time: 3 minutesBlocking and dilution buffer: 5% NFDM/TBST.
The expression profile observed is consistent with what has been described in the literature (PMID: 9657756).
-
![Immunoprecipitation - Anti-TNF alpha antibody [EPR22598-212] (ab255275)](https://www.abcam.com/ps/products/255/ab255275/Images/ab255275-347218-anti-tnf-antibody-epr22598-212-immunoprecipitation-thp1-human.jpg)
Immunoprecipitation - Anti-TNF alpha antibody [EPR22598-212] (ab255275)TNF alpha was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) (treated with 80nM TPA overnight, replaced the culture medium with 100 ng/ml Lipopolysaccharides (LPS) for 6 hours with addition of 300ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate with ab255275 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab255275 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.
Lane 1: THP-1 (treated as above) whole cell lysate 10 μg (Input).
Lane 2: ab255275 IP in THP-1 (treated as avove) whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255275 in THP-1 (treated as above) whole cell lysate.Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds. -
![Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)](https://www.abcam.com/ps/products/255/ab255275/Images/ab255275-347219-anti-tnf-antibody-epr22598-212-western-blot-raw2647-mouse.jpg)
Western blot - Anti-TNF alpha antibody [EPR22598-212] (ab255275)All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution
Lane 1 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 (treated with 100 ng/ml lipopolysaccharide (LPS) for 6 hours with addition of 300 ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 25 kDa
Observed band size: 25 kDa
Exposure time: 3 minutesBlocking and dilution buffer: 5% NFDM/TBST.
The expression profile observed is consistent with what has been described in the literature (PMID: 9657756).
-
![Anti-TNF alpha antibody [EPR22598-212] (ab255275)](https://www.abcam.com/ps/products/255/ab255275/Images/ab255275-5-benefits-of-recombinant-antibodies.png)
Anti-TNF alpha antibody [EPR22598-212] (ab255275)
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
-
SDS download
-
Datasheet download
Certificate of Compliance
References (1)
ab255275 has been referenced in 1 publication.
- Li M et al. CircPTK2-miR-181c-5p-HMGB1: a new regulatory pathway for microglia activation and hippocampal neuronal apoptosis induced by sepsis. Mol Med 27:45 (2021). PubMed: 33952191