Cell lysates collected in RIPA buffer (supplemented with 1 mM PMSF, 1× protease inhibitor) were separated by SDS–PAGE, transferred to a PVDF membrane (Immobilon-P, Merck Millipore Ltd), and incubated with primary antibodies (1:1000) at 4 °C overnight. After subsequent staining with HRP-conjugated secondary antibodies (1:10000 for anti-rabbit and 1:3000 for anti-mouse antibodies; 4 °C; overnight), the signal was developed using the ECL Star kit (Euroclone SpA, according to the instruction of the manufacturer using the BioRad ChemidocTM imaging system (Image LabTM touch software, BioRad). β2-actin was used as the loading control. Western blot experiments were performed with anti-ATG5, anti-PU.1, anti-Beclin1, anti-IRF1 (D5F5U; 9G7; D40C5; D5E4, Cell Signaling Technology), anti-LC3I/LCRII (MBL-PM036, MBL International), anti-CRLS1 (PA5–25338, Invitrogen), anti-LPCAT1, anti-PGS1 (PA5–26318; PA5–43247, Invitrogen) and anti-β2 actin (SC-47778, Santa Cruz) antibodies.
Ecl star kit
Manufactured by Euroclone
The ECL star kit is a laboratory equipment product designed for chemiluminescent detection. It provides a solution for the detection and analysis of proteins and nucleic acids in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (3 mentions)
Tween 20, by Merck Group (2 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Anti p27 kip1, by Abcam (1 mentions)
Anti phospho her2 erbb2 tyr1248, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Rabbit anti gapdh, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 protocols using ecl star kit
1
Western Blot Analysis of Autophagy Proteins
2
Western Blot Analysis of HER2 Signaling
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A 20 μg protein aliquot was resuspended in Laemmli buffer, resolved on polyacrylamide gels under reducing conditions and transferred to polyvinylidine difluoride membranes (Immobilon-P, EMD Millipore Corporation). Membranes were blocked with 5% skim milk or 5% BSA in Tris buffer saline (TBS) with 0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature, and incubated with appropriate primary antibodies: anti-phospho-HER2/ErbB2 Tyr1248 (Cell Signaling Technology, Inc.), anti-HER2/ErbB2 (clone 29D8, Cell Signaling Technology, Inc.), anti-p27 KIP1 (Abcam), or anti-α-tubulin (Sigma-Aldrich). Antibodies conjugated to horseradish peroxidase (Abcam) were used as secondary antibodies, and chemiluminescence reaction was developed with the ECL star kit (Euroclone). Densitometric analysis of protein bands was performed with ImageJ software.
3
Quantification of MAdCAM-1 by Western Blot
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Frozen tissues were weighted, homogenized with potter (Glas-Col homogenizer) in 20% w/v Triton lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 10% Glycerol and 1% Triton X-100), containing 4% Protease Inhibitor Cocktail (Sigma-Aldrich S.r.l., Milan, IT), and cleared at 17,100 ×g for 20 min at 4 °C. A 40 µg aliquot of protein was resuspended in Laemmli buffer, resolved on polyacrylamide gels under reducing conditions and transferred to polyvinylidene difluoride membranes (Immobilon-P, EMD Millipore Corporation, Billerica, MA-US). Membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) in Tris buffer saline with 0.1 % Tween-20 (Sigma-Aldrich) for 1 h at RT, and incubated with rat anti-MAdCAM-1 antibody (MECA-367, LifeSpan BioSciences, Seattle, WA) or with rabbit anti-GAPDH (Sigma-Aldrich). Antibodies conjugated to horseradish peroxidase (Abcam) were used as secondary antibodies, and the reaction was developed with the ECL star kit (Euroclone). Densitometric analysis of protein bands was performed with ImageJ software.
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