Cells were lysed in RIPA buffer (10mM Tris-HCl [pH 8.0], 150mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad). SDS–PAGE and protein transfer were performed using Mini Gel Tank and Mini Blot Module (Life Technologies). Immunoblotting was detected using near-infrared fluorescence (LI-COR) and the Odyssey CLx imager (LI-COR). Quantitative analysis of immunoblots was performed using Image Studio Lite software (LI-COR). The following primary antibodies were used: EREG (1:1000) [Cell Signaling Technologies (CST), 12048], HB-EGF (1:10000) (Abcam, ab185555), p-EGFR (Y1068) (1:1000) (CST, 3777), EGFR (1:1000) (CST, 4267), p-PAK1 (S199/204)/p-PAK2 (S192/197) (1:500) (CST, 2605), PAK1 (1:300) (Santa Cruz, sc-882), p-Erk1/2 (T202/Y204) (1:2000) (CST, 4370), Erk1/2 (1:1000) (CST, 4695), p-Akt (S473) (1:1500) (CST, 4060), pan-Akt (1:1000) (CST, 4691), α-tubulin (1:10000) (Sigma, T6074), and β-actin (1:20000) (Sigma, A1978). To analyze phosphorylation of proteins, membranes were probed with phospho-specific antibodies first, stripped with NewBlot IR Stripping Buffer (LI-COR) and then reprobed with pan antibodies.
Newblot ir stripping buffer
Manufactured by LI COR
NewBlot IR Stripping Buffer is a solution designed to remove primary and secondary antibodies from Western blot membranes. It is used to prepare the membrane for re-probing with new antibodies, allowing for the detection of multiple target proteins on the same blot.
Automatically generated - may contain errors
Lab products found in correlation
Pakt s473, by Merck Group (1 mentions)
P egfr, by Abcam (1 mentions)
Mini blot module, by Thermo Fisher Scientific (1 mentions)
Pan akt, by Merck Group (1 mentions)
Image studio lite software, by LI COR (1 mentions)
α tubulin, by Merck Group (1 mentions)
Near infrared fluorescence, by LI COR (1 mentions)
Hb egf, by Abcam (1 mentions)
Erk1 2, by Merck Group (1 mentions)
Mini gel tank, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Santa Cruz Biotechnology (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
β actin, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
Trueblack antibody dilutent, by Biotium (1 mentions)
Immobilon fl, by Merck Group (1 mentions)
2 protocols using newblot ir stripping buffer
1
Protein Extraction and Western Blot Analysis
2
Fluorescence-Based Western Blotting Protocol
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For fluorescence Western blotting, samples were analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride Immobilon FL (Millipore). Membranes were dried for at least 1 h, rehydrated in methanol, and stained for total protein (LI-COR REVERT Total Protein Stain). Following imaging of the total protein, membranes were destained, blocked for 1 h in TrueBlack Blocking Buffer (23013; Biotium), and incubated overnight at 4°C with primary antibodies diluted in TrueBlack Antibody Dilutent (23013; Biotium) with 0.2% Tween-20. Membranes were washed four times for 5 min in 1×TBS washing solution (50 mM Tris-HCl [pH 7.4], 274 mM NaCl, 9 mM KCl, 0.1% Tween-20), incubated in secondary antibodies diluted in TrueBlack Antibody Dilutent (23013; Biotium) with 0.2% Tween-20 and 0.01% SDS for 1 h, and again washed four times for 5 min in washing solution. Membranes were immediately imaged using an Odyssey CLx Infrared Imaging System (LI-COR). In a limited number of cases, the membrane was stripped using NewBlot IR Stripping Buffer (928–40028; LI-COR) according to manufacturer’s instructions. Band intensity was measured in the LI-COR Image Studio application.
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