HCCC-9810 and HUCCT1 cells were lysed and total protein was extracted. BCA Protein Assay Kit (HyClone-Pierce) was used to detect protein concentration. Then 20 µg protein was used for SDS-PAGE. The proteins were then transferred to PVDF membranes, which were then blocked with 1 × TBST solution containing 5% skim milk for 1 h at room temperature. PVDF membranes were incubated with primary and secondary antibodies for 1 h at room temperature, and then washed three times with 1 × TBST for 10 min each time. Immobilon Western Chemiluminescent HRP Substrote Kit (Millipore) was used to develop protein color, and then chemiluminescence was performed with a chemiluminescence imager (GE), and the protein bands were photographed. Information on primary and secondary antibodies used in western blot assay was provided in Supplementary Table 1 .
Immobilon western chemiluminescent hrp substrote kit
Manufactured by Merck Group
Sourced in United States
The Immobilon Western Chemiluminescent HRP Substrate kit is a laboratory product designed to detect and quantify proteins on western blots. The kit contains the necessary reagents for chemiluminescent detection of horseradish peroxidase (HRP)-conjugated secondary antibodies.
Automatically generated - may contain errors
6 protocols using immobilon western chemiluminescent hrp substrote kit
1
Western Blot Analysis of Protein Extracts
2
Western Blot Analysis of Gastric Cancer Cells
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The gastric cancer cells were lysed using 1 X lysis buffer and the total protein was extracted. BCA Protein Assay Kit (23225, HyClone-Pierce) was used to detect protein concentration. 20 μg of protein was taken for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to Polyvinylidene Fluoride (PVDF) membranes. PVDF membranes were blocked in 1 X TBST solution containing 5% skimmed milk for 1 h at room temperature, and incubated with the diluted primary antibodies for 2 h at room temperature. 1 X TBST solution was employed to wash the membranes 3 times, 10 min each time. PVDF membranes were incubated in dilute secondary antibodies for 1 h at room temperature. After washing membranes 3 times, the chemiluminescence was carried out by using the immobilon Western chemiluminescent HRP Substrote Kit (RPN2232, Millipore), and the chemiluminescence imaging system (AI600, GE) was used for imaging. GAPDH was used as an internal reference. The primary and secondary antibodies involved in this experiment were provided in Supplementary Table 1 .
3
Protein Expression Detection via SDS-PAGE
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After the cells BT549 and MDA‐MB‐231 had been lysed, the protein was obtained, and the protein quality was detected using BCA protein detection kit (HyClone‐Pierce). The 10‐µg protein was separated by SDS‐PAGE (Invitrogen), transferred to the PVDF membrane, and then sealed with TBST solution. Subsequently, the protein was first incubated with primary antibody at 37°C for 2 hours (Table S1 ), and then with secondary antibody at 4°C overnight. Finally, the Millipore Immobilon Western Chemiluminescent HRP Substrote kit (Millipore, Cat. No. RPN2232) was used for color rendering and chemiluminescent imager (GE, Cat. No. AI600) observation.
4
Western Blot Analysis of Cellular Proteins
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The total proteins of MNNG/HOS and U-2OS were extracted with cell lysate, and detected by BCA protein detection kit (HyClone-Pierce). The 10-µg protein was separated by SDS-PAGE (Invitrogen) and transferred to the PVDF membrane, then sealed at room temperature for 1 h with TBST solution. After that, the membrane was first incubated with primary antibodies (Additional file 6 : Table S1) overnight at 4 °C, and with HRP-conjugated goat anti-rabbit IgG for another 2 h at room temperature (The antibody information was shown in Additional file 6 : Table S1). Finally, Millipore Immobilon Western Chemiluminescent HRP Substrote kit (Millipore, Cat. No. RPN2232) was used for color rendering and Chemiluminescent imager (GE, Cat. No. AI600) observation.
5
Protein Expression Analysis and Co-IP
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After the U-CH1 and MUG-Chor1 cells were lysed, the protein was obtained and the concentration was measured by BCA protein detection kit (Thermo Fisher Scientific, California, USA). The protein with equal amounts was subjected to SDS-PAGE, transferred to nitrocellulose membranes and incubated with primary antibodies (Table S2 ) overnight at 4 °C. After washed by TBST, the membranes were probed with secondary antibodies. Finally, Millipore Immobilon Western Chemiluminescent HRP Substrote kit (Millipore, Cat. No. RPN2232) was used to color rendering and Chemiluminescent imager (GE, Cat. No. AI600) observation. The protein–protein interaction was analyzed by Co-IP assay and the experimental procedures were performed as previously described [27 (link)].
6
Western Blot Analysis of Melanoma Cells
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The melanoma cells infected with lentivirus were collected and the total protein was extracted. The protein concentration was determined by bicinchoninic acid (BCA) Protein Assay Kit (23225, HyClone-Pierce, UT, USA). The total protein was separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and then transferred to Polyvinylidene fluoride (PVDF) membrane (Millipore Life Science, MA, USA). 1× Tris Buffered Saline with Tween (TBST) solution with 5% skimmed milk was used to seal the membrane for 1 h at room temperature. The PVDF membrane was incubated with the primary antibody overnight at 4 °C, and incubated with the secondary antibody for 1 h at room temperature. 1× TBST solution was used to wash the membrane for 10 min, three times in total. Under the instructions of Immobilon Western Chemiluminescent HRP Substrote Kit (Millipore), a chemiluminescence imager (GE Healthcare Life Sciences, Boston, MA, USA) was used for chemiluminescence and imaging. The antibodies are listed in Supplementary Table 1. This experiment was repeated three times independently.
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