Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free

• IP

Unknown

Immunoprecipitation - Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free (AB222500)

TNF alpha was immunoprecipitated from 0.35 mg of TPA pretreated THP-1 (Human monocytic leukemia cell line) whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours with ab183218 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab183218 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : TPA pretreated THP-1 whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours 10μg (Input).

Lane 2 : ab183218 IP in TPA pretreated THP-1 whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours.

Lane 3 : Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab183218 in TPA pretreated THP-1 whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183218).

All lanes:

Immunoprecipitation - Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free (ab222500)

Predicted band size: 25 kDa

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• ELISA

Unknown

ELISA - Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free (AB222500)

ELISA using ab183218 between 0.1 and 1000 ng/ml to detect human TNF alpha peptide. Secondary used was Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) 1/2000.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183218).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free (AB222500)

Immunocytochemistry/ Immunofluorescence analysis of RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 7 h and 1μg/ml BFA for the last 3h cells labeling TNF alpha with purified ab183218 at 1/5000 dilution (0.09 μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488 ab150077) was used as the secondary antibody at 1/1000 dilution (2 μg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS BSA glycerol and sodium azide (ab183218).

• WB

Lab

Western blot - Anti-TNF alpha antibody [EPR19147] - Low endotoxin, Azide free (AB222500)

This data was developed using the same antibody clone in a different buffer formulation (ab183218).

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 55 kDa.

ab183218 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 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.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR19147] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr19147-ab183218'>ab183218</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 2:

Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 2:

Western blot - Human TNF knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-tnf-knockout-thp-1-cell-line-ab273761'>ab273761</a>)

Lane 3:

TNF alpha knockout THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 4:

TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 5:

U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 6:

U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Predicted band size: 25 kDa

Observed band size: 26 kDa

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