Human TNF knockout THP-1 cell lysate (ab275507)
Overview
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Product name
Human TNF knockout THP-1 cell lysate
See all TNF alpha kits -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9.
Treatments:
Human TNF knockout THP-1 cell lysate - Brefeldin A (5 µg/ml, 6h)
Human wild-type THP-1 cell lysate - Brefeldin A (5 µg/ml, 6h)
Human TNF knockout THP-1 cell lysate - LPS (1 µg/ml, 16h) and Brefeldin A (5 µg/ml, during the last 6h of LPS treatment)
Human wild-type THP-1 cell lysate - LPS (1 µg/ml, 16h) and Brefeldin A (5 µg/ml, during the last 6h of LPS treatment) -
Parental Cell Line
THP-1 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 52 bp deletion in exon 4 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab277319 - Human TNF knockout THP-1 cell lysate - Brefeldin A treated 1 x 100µg ab277320 - Human TNF knockout THP-1 cell lysate - LPS + Brefeldin A treated 1 x 100µg ab277492 - Human wild-type THP-1 cell lysate - Brefeldin A treated 1 x 100µg ab277321 - Human wild-type THP-1 cell lysate - LPS + Brefeldin A treated 1 x 100µg -
Research areas
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Cell type
acute monocytic leukemia -
Disease
Acute Monocytic Leukemia -
Gender
Male
Target
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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
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Alternative names
- APC1
- APC1 protein
- Cachectin
see all
Associated products
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KO cell lines
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Related Products
- Anti-TNF alpha antibody [EPR19147] (ab183218)
- Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free (ab222500)
- Anti-TNF alpha antibody [EPR22598-212] (ab255275)
- Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)
- Anti-TNF alpha antibody [EPR19147] - BSA and Azide free (ab271939)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab275507 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 |
Use at an assay dependent concentration. Predicted molecular weight: 25 kDa.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 25 kDa. |
Images
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Western blot - Human TNF knockout THP-1 cell lysate (ab275507)
Lane 1: Wild-type THP-1 control: Brefeldin A (5 μg/mL, 4 h) cell lysate 30 μg
Lane 2: Wild-type treated THP-1: LPS (100 ng/mL, 16 h), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
Lane 3: TNF alpha knockout THP-1 control: Brefeldin A (5 μg/mL, 4 h) cell lysate 30 μg
Lane 4: TNF alpha knockout THP-1 treated: LPS (100 ng/mL, 16 h), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
Lane 5: U937 control: PMA (10 mM, 2 days), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
Lane 6: U937 treated: PMA (10 mM, 2 days), LPS (1 μg/mL, last 16 h), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
This Western blot image is a comparison between ab215188 and ab183218 tested under the same conditions. While ab215188 is suitable for WB for some samples, ab183218 was found to be more sensitive.False colour image of Western blot: Anti-TNF alpha antibody [EPR20972] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab215188 was shown to bind specifically to TNF alpha. A band was observed at 27 kDa in treated U937 cell lysates with no signal observed at this size without treatment. No signal was observed in wild-type THP-1 cell lysates or in TNF knockout cell line ab273761 (knockout cell lysate ab275507) with ab215188. However, a band was observed at 27 kDa in treated wild-type THP-1 cell lysates with ab183218. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution. -
Western blot -Human TNF knockout THP-1 cell lysate
(ab275507)Lane 1: Wild-type THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 2: Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 3: TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 4: TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 5: U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 6: U937 PMA treated (10 mM, 2 days) and LPS treated (1 ug/ml, 16 h) plus Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
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 55kDa.
ab255275 was shown to react with TNF alpha in wild-type THP-1 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. -
Western blot -Human TNF knockout THP-1 cell lysate
(ab275507)Lane 1: Wild-type THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 2: Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 3: TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 4: TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 5: U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 6: U937 PMA treated (10 mM, 2 days) and LPS treated (1 ug/ml, 16 h) plus Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lanes 1 - 6:Merged signal (red and green). Green - ab183218 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab183218 was shown to react with TNF alpha in wild-type THP-1 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 ab183218 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. -
Sanger Sequencing - Human TNF knockout THP-1 cell lysateHomozygous: 52 bp deletion in exon 4
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
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SDS download
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Datasheet download
References (0)
ab275507 has not yet been referenced specifically in any publications.