0.01M TBS (pH7.4) with 1% rAlbumin, 0.02% Proclin300 and 50% Glycerol.
Form:
Liquid
Target:
MAP3K5
Application Dilute:
WB=1:500-2000, Flow-Cyt=1µg /test
Application Notes:
Modification: Phosphorylated
Western blot of mouse brain tissue using ASK1 (phospho-Thr845) antibody.
Blank control (blue): Hela (fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice). Primary Antibody: Rabbit Anti-phospho-ASK1 (Thr845) antibody (orb6390), dilution: 0.2 µg in 100 µl 1X PBS containing 0.5% BSA, Isotype Control Antibody: Rabbit IgG (orange), used under the same conditions, Secondary Antibody: Goat anti-rabbit IgG-PE (white blue), dilution: 1:200 in 1X PBS containing 0.5% BSA.
Blank control: Mouse spleen. Primary Antibody (green line): Rabbit Anti-phospho-RelB (Ser551) antibody (orb6390), 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 90% ice-cold methanol for 20 min at -20C. 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: U937. Primary Antibody (green line): Rabbit Anti-phospho-ASK1 (Thr845) antibody (orb6390), dilution: 1 ug/Test, Secondary Antibody: Goat anti-rabbit IgG-FITC, dilution: 0.5 ug/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 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: Brain (Rat) Lysate at 30 ug, Lane 2: Heart (Rat) Lysate at 30 ug, Primary: Anti-phospho-ASK1 (Thr845) (orb6390) at 1:200 dilution, Secondary: HRP conjugated Goat-Anti-Rabbit IgG (orb572747) at 1:3000 dilution, Predicted band size: 155kD, Observed band size: 155kD.
Sample: Lane 8: Cerebrum (Rat) Lysate at 40 ug, Lane 9: Stomach (Rat) Lysate at 40 ug, Primary: Anti-phospho-ASK1 (Thr845) (orb6390) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 154 kD, Observed band size: 154 kD.
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