Protein extraction was done using the standard protocol as previously described (Liger et al. 2011 (link)) for FLAG-tagged proteins or the Kushnirov extraction method (Kushnirov 2000 (link)) for myc-tagged proteins. Proteins from soluble fractions were resolved by SDS-PAGE on a 15% gel and transferred onto Protran nitrocellulose membrane (Whatman). Detection of myc-tagged proteins was performed using mouse 9E10 anti-myc as primary antibody (1/3000 dilution, Sigma-Aldrich) and goat anti-mouse HRP-conjugated IgG as secondary antibody (1/5000 dilution) (Sigma-Aldrich). Detection of FLAG-tagged proteins was performed using mouse anti-FLAG M2-peroxidase (HRP) (1/10 000 dilution) (Sigma-Aldrich). Immunoblots were revealed using ECL kit SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher).
Ecl kit supersignal west pico chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
The ECL kit SuperSignal West Pico Chemiluminescent Substrate is a laboratory equipment product that is used for the detection of proteins in Western blot analysis. It provides a sensitive chemiluminescent signal for visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti myc 9e10, by Merck Group (1 mentions)
Protran nitrocellulose membrane, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
Protran, by Cytiva (1 mentions)
Anti actin clone c4, by Merck Group (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti pgsk3α β, by Cell Signaling Technology (1 mentions)
2 protocols using ecl kit supersignal west pico chemiluminescent substrate
1
Western Blot Analysis of Tagged Proteins
2
Immunoblotting analysis of T cells and Jurkat cells
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Primary T cells (1 × 106) and Jurkat cells (3 × 106) were lysed in lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100) in the presence of Protease Inhibitor Cocktail Set III (Cal BioChem) and 0.2 mg Na orthovanadate/mL. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane (GE Healtcare) for immunoblotting analysis. Immunoblots were performed using the following primary antibodies: anti-Rai (BD, #610643), anti-pGSK3α/β (Cell Signaling, #8566), anti-pAkt (Cell Signaling, #9271), anti-actin (clone C4, Millipore, #MAB1501) and peroxidase-labeled secondary antibodies (anti-rabbit cod: 111-035-144 and anti-mouse cod:115-035-146 Jackson ImmunoResearch Laboratories). Labeled Abs were detected using the ECL kit SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific) and scanned immunoblots were quantified using the ImageJ software (version 1.43r).
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