According to our previous study [15 (link)], 20 µg of total protein was loaded for SDS-PAGE electrophoresis and transferred to the PVDF membrane at a voltage of 110 V (90 min). After that, the membrane was blocked in 5% skimmed milk for 1 h, and incubated with primary antibodies (anti Flag: #2064, Dia-An Biotechnology, 1:3000; anti-NF-κB: #8242, Cell Signaling Technology, 1:1000; anti-phos NF-κB: #3033, Cell Signaling Technology, 1:1000; anti-ERK: #4695, Cell Signaling Technology, 1:2000; anti-phos ERK: #4370, Cell Signaling Technology, 1:3000; anti-GAPDH: sc-365,062, Santa Cruz Biotechnology, 1:2000) at 4 oC overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. The protein signals were detected with ECL kit. GAPDH was used as a loading control.
Anti phos erk
Manufactured by Cell Signaling Technology
Anti-phos ERK is a primary antibody that recognizes the phosphorylated form of extracellular signal-regulated kinase (ERK) proteins. It can be used to detect and quantify the activation of the ERK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Anti p100 p52, by Cell Signaling Technology (1 mentions)
2 protocols using anti phos erk
1
Western Blot Analysis of Protein Expression
2
Thalidomide Modulates NF-κB and ERK Signaling
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BMOL T cells were exposed to thalidomide C1 as indicated above and lysates were prepared in DISC lysis buffer as described previously [22] . Its effect on NFκB and ERK signalling pathways were assessed by Western Blot using the following antibodies; anti-ERK (#4696), anti-Phos-ERK (#9101), anti-p100/p52 (#4882), anti-Phospho-p65 (#3033, Cell Signaling Technology) and anti-β-actin (A1978, Sigma-Aldrich). Briefly the lysates were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (#10600018, GE Healthcare) for blotting. Membranes were incubated with the primary antibodies overnight followed by HRP conjugated secondary antibodies for 1hour. All antibodies were diluted in in 5% skim milk powder in Tris Buffered Saline with 0.1% Tween (TBS-T).
The chemiluminescent signal was developed using the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck Millipore) and captured using a BioRad ChemiDoc™ MP System.
The chemiluminescent signal was developed using the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck Millipore) and captured using a BioRad ChemiDoc™ MP System.
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