0.01M TBS (pH7.4) with 1% rAlbumin, 0.02% Proclin300 and 50% Glycerol.
Form:
Liquid
Target:
TNF
Application Dilute:
ICC/IF=1:100-500, Flow-Cyt=1ug/Test
Blank control: Raw264.7. Primary Antibody (green line): Rabbit Anti-TNF alpha antibody (orb317993), dilution: 1 ug/Test, Secondary Antibody: Goat anti-rabbit IgG-FITC, dilution: 0.5 ug/Test. Protocol, The cells were treated with LPS (1 ug/ml, 18 hr/6 hr) and Brefeldin A (300 ng/ml, last 3 hr of stimulation). The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
Blank control: Raw264.7. Primary Antibody (green line): Rabbit Anti-TNF alpha antibody (orb317993), dilution: 2 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-AF488, dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
Blank control: THP-1. Primary Antibody (green line): Rabbit Anti-TNF alpha antibody (orb317993), dilution: 1 ug/Test, Secondary Antibody: Goat anti-rabbit IgG-FITC, dilution: 0.5 ug/Test. Protocol, The cells were treated with TPA (80 nM, overnight) and then treated with LPS (1 ug/ml, 18 hr/6 hr) and Brefeldin A (300 ng/ml, last 3 hr of stimulation). The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
Sample: Lane 1: A431 (Human) Cell Lysate at 30 ug, Lane 2: Hela (Human) Cell Lysate at 30 ug, Primary: Anti-TNF alpha (orb317993) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 36/17/26 kD, Observed band size: 36 kD.
Western blot analysis of Mouse Lymph Lysate using TNF alpha antibody.
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