Human TNF-alpha Antibody (6401)

Cytotoxicity Induced by TNF‑ alpha and Neutralization by Human TNF‑ alpha Antibody.

Recombinant Human TNF-a (Catalog # 210-TA) induces cytotoxicity in the L-929 mouse fibroblast cell line in a dose-dependent manner (orange line). Cytotoxicity elicited by Recombinant Human TNF-a (0.25 ng/mL) is neutralized (green line) by increasing concentrations of Human TNF-a Monoclonal Antibody (Catalog # MAB2101). The ND₅₀ is typically 0.015-0.06 µg/mL in the presence of the metabolic inhibitor actinomycin D (1 µg/mL).

Detection of TNF‑ alpha in Human PBMC Monocytes by Flow Cytometry.

Human PBMCs were treated with 1μg/ml LPS for 4 hours then stained with (A) Mouse Anti-Human TNF-a Membrane Form Monoclonal Antibody (Catalog # MAB2101) or (B) isotype control antibody (MAB002) followed by anti-Mouse IgG PE-conjugated Secondary Antibody (F0102B) and Mouse anti-Human CD14 APC-conjugated Monoclonal Antibody (FAB3832A). Staining was perfomed using our Staining Membrane-associated Proteins protocol.

Detection of Insect TNF-alpha by Western Blot

The pre-integrated TMD docks to the cytosolic tip of the lateral gate.(A) Co-immunoprecipitation of recombinant FLAG-Sec61 alpha and Sec61 gamma. Sf21 insect cell microsomes were solubilized with 1% Deoxy BigChap (DBC) and subjected to anti-FLAG immunoprecipitation (IP). Eluted samples (Sf21) were analyzed by immunoblotting next to a sample of canine microsomal ER membranes (CRM). (B) Comparison of the relative CT7 photo-crosslinking efficiency of canine rough microsomes (CRM) and Sf21 microsomes containing wild-type FLAG-Sec61 alpha / gamma complex. CT7 crosslinking was performed as before. The relative crosslinking efficiency of recombinant Sec61 alpha was normalized to the native protein. (C) BMH crosslinking reactions using recombinant FLAG-Sec61 alpha / gamma complex. Cys49 TNF alpha 126-mers were prepared in the presence of Sf21 microsomes, and BMH crosslinking reactions were conducted as described previously. Reactions were either analyzed directly (Total) or after denaturing IP with anti-FLAG affinity resin. IP with the anti-HA affinity resin served as a specificity control. (D) Western blots of Sf21 microsomes co-expressing either wild-type or mutant FLAG-Sec61 alpha / gamma. Insect cell microsomes from cells infected with a baculovirus that lacked Sec61 genes (control virus) served as a control. (E) CT7 crosslinking of mutant Sec61 alpha / gamma complexes containing the indicated cysteine substitutions. The region of the gel corresponding to FLAG-Sec61 alpha is shown. Detection of photo-affinity labeling that is competed by CT9 indicates that the mutant Sec61 alpha / gamma complex is properly folded.DOI:https://dx.doi.org/10.7554/eLife.01483.010 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/01483), licensed under a CC-BY license. Not internally tested by R&D Systems.