Anti-AP-1 antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Homogenates were diluted to 4 mg/ml in loading buffer (62.5 mM Tris, pH 6.8, 1.0% SDS, 0.01% bromophenol blue, 15.0% glycerol, and 5% β-mercaptoethanol). Forty micrograms of protein were loaded into each well in 4–20% precast gradient gel (Bio-Rad, Hercules, CA), and proteins were separated at room temperature for 10 min at 60 V, followed by 60 min at 120 V. Afterward, proteins were transferred (60 min, 100 V) to a nitrocellulose membrane (Bio-Rad, Hercules, CA) with a pore diameter of 0.2 μm. Membranes were blocked for 1 h in 5% milk in Tris-buffered saline containing 0.2% Tween 20 (TTBS). Membranes with protein from whole homogenate were incubated with each of the following: heat shock protein (HSP) 72 (primary 1:1,000, cat. no. NB120-2788, Novus Biological, Littleton, CO; secondary 1:5,000), HSP27 (primary 1:1,000, cat. no. ADI-SPA-800, ENZO Life Sciences, Farmingdale, NY; secondary 1:2,000), phospho-HSP27 (primary 1:1,000, cat. no. 2406, Cell Signaling Technology, Danvers, MA; secondary 1:1,500), HSP90 (primary 1:1,000, cat. no. 4877, Cell Signaling Technology; secondary 1:1,500), TNF-α (primary 1:400, cat. no. ab6671, Abcam, Cambridge, MA; secondary 1:5,000), NF-κB (primary 1:5,000, cat. no. ab7970, Abcam, secondary 1:5,000), phospho-NF-κB p65 (primary 1:1,000, cat. no. MA5-15160, Thermo Scientific, Rockford, IL; secondary 1:2,000), inhibitory κB kinase-α (IKB-α) (primary 1:1,000, cat. no. SC-371, Santa Cruz Biotechnology, Dallas, TX; secondary 1:2,000), inhibitory κB kinase-α (IKK-α) (primary 1:1,000, cat. no. SAB4500258, Sigma Aldrich, St. Louis, MO; secondary 1:5,000), SAPK/JNK (primary 1:1,000, cat. no. 9252s, Cell Signaling Technology, secondary 1:2,000) or phospho-SAPK/JNK (primary 1:1,000, cat. no. 4668s, Cell Signaling Technology; secondary 1:2,000), IL-1β (primary 1:1,000, cat. no. 12703, Cell Signaling Technology; secondary 1:1,000), IL-2 (primary 1:1,000, cat. no. 12703, Cell Signaling Technology; secondary 1:1,000), IL-6 (primary 1:1,000, cat. no. ab6672, Abcam; secondary 1:2,000) overnight at 4°C. Membranes with protein from the nuclear fraction were incubated with antibodies to NF-κB (primary 1:5,000, cat. no. ab7970, Abcam; secondary 1:5,000), phospho-NF-κB p65 (primary 1:1,000, cat. no. 3033, Cell Signaling Technology; secondary 1:2,000), AP-1 (primary 1:1,000, cat. no. A5968, Sigma Aldrich; secondary 1:3,000), heat shock factor (HSF)-1 (primary 1:1,000, cat. no. 4356; Cell Signaling Technology; secondary 1:3,000) or phospho-HSF-1 (primary 1:5,000; cat. no. ab76076, Abcam; secondary 1:3,000) overnight at 4°C.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting C-Jun using Anti-AP-1 antibody produced in rabbit from Merck Millipore.

Manufacturer protocol

Download the product protocol from Merck Millipore for Anti-AP-1 antibody produced in rabbit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting C-Jun using Anti-AP-1 antibody produced in rabbit from Merck Millipore. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.