Total proteins were extracted from cells and tumor tissues in radioimmunoprecipitation assay buffer. The cells were collected, washed three times with precooled PBS, and centrifuged at 1500 rpm for 3 min. An appropriate volume of cell lysate was added, and the cells were lysed on ice for 30 min. The supernatant was centrifuged at 1200 rpm at 4°C for 30 min and stored at –80°C. After assaying the protein concentration, samples were added to 4× sodium dodecyl sulfate loading buffer, boiled for 5 to 10 min, and centrifuged at 12,000 × g for 1 min. Proteins (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). Primary antibodies against GLUT-1 (Abcam), beclin-1, LC3 (Proteintech), Atg7, Atg5, HIF-1α and β-actin (Abcam) were added and incubated at 4°C overnight; β-actin served as the internal control. After washing three times with Tris-buffered saline/Tween 20, the secondary antibodies were added for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence assay kit (Beyotime Biotech) and analyzed semi-quantitatively using the ChemiDoc XRS+ System (Bio-Rad).
Enhanced chemiluminescence assay kit
Manufactured by Beyotime
Sourced in China
The Enhanced Chemiluminescence Assay Kit is a laboratory equipment designed to detect and quantify the presence of specific proteins in a sample. It utilizes a chemiluminescent reaction to generate a luminescent signal, which can be measured to determine the target protein concentration.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (3 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Hif 1α, by Abcam (1 mentions)
Glut1, by Abcam (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Anti p stat1, by Cell Signaling Technology (1 mentions)
P smad, by Abcam (1 mentions)
Anti socs1, by Cell Signaling Technology (1 mentions)
Hcv core, by Abcam (1 mentions)
P jnk, by Abcam (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Xrs imaging system, by Bio-Rad (1 mentions)
Af1891, by Beyotime (1 mentions)
Af1051, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab156872, by Abcam (1 mentions)
Ab3366, by Abcam (1 mentions)
7 protocols using enhanced chemiluminescence assay kit
1
Protein Extraction and Western Blot Analysis
2
Regulation of SOCS1 and STAT1 in THP-1 Cells
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THP-1 cells were transfected with 100 pmol mimics-155 and negative control. The cells were lysed on ice in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% deoxycholic acid, 0.1% SDS, 1% Triton X-100). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Then, membranes were blocked with 5% milk-0.5% Tween-20 in Tris-buffered saline and probed with the anti-SOCS1 and anti-p-STAT1 (Cell Signaling Technology (CST), Shanghai, China) at 4 °C overnight. Subsequently, the membrane was incubated with horseradish peroxide-conjugated secondary antibody (CST). The immunoreactive bands were detected using an enhanced chemiluminescence assay kit (Beyotime Biotechnology, Shanghai, China).
THP-1 cells were divided into four groups: the control group incubated in PBS, and the experimental group treated with LPS (Sigma-Aldrich, MO), HCV CORE, or HCV NS5 (Abcam, Shanghai, China) antigens, respectively. After 1 day of culturing, the cells were lysed and protein levels were assessed by Western blot. The changes in cell signaling were detected after the stimulation of each antigen, and the phosphorylation of p65, smad, p38, and JNK proteins was detected. The applied antibodies were p65, p-smad, p-p38, and p-JNK (Abcam).
THP-1 cells were divided into four groups: the control group incubated in PBS, and the experimental group treated with LPS (Sigma-Aldrich, MO), HCV CORE, or HCV NS5 (Abcam, Shanghai, China) antigens, respectively. After 1 day of culturing, the cells were lysed and protein levels were assessed by Western blot. The changes in cell signaling were detected after the stimulation of each antigen, and the phosphorylation of p65, smad, p38, and JNK proteins was detected. The applied antibodies were p65, p-smad, p-p38, and p-JNK (Abcam).
3
Western Blot Analysis of BRCA1 and HER2
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The tumor tissues were lysed in lysis buffer and then centrifuged at 15,000 rpm for 15 min at 4°C. The protein concentration was determined using a bicinchoninic acid (BCA) kit (Beyotime, China). A total of 50 μg of protein was separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, USA). The membranes were blocked for 1 h at 26°C with 5% bovine serum albumin containing 0.1% Tween-20, incubated with the primary antibodies (breast cancer gene 1 (BRCA1) and HER2, Beyotime, China; 1 : 1000) overnight at 4°C. Then, the membranes were washed with Tris-Buffered Saline with Tween-20 (TBST) three times and incubated with the corresponding secondary antibody (1 : 5000) at 37°C for 2 h. The membranes were then washed again and the proteins were detected using an enhanced chemiluminescence assay kit (Beyotime, China), followed by image capturing using the BioRad XRS + imaging system (BioRad, USA).
4
Tumor Protein Expression Analysis
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The expressions of EGF (Abcam, ab184265, 1 : 1000), VEGF (Abcam, ab32152, 1 : 1000), NF-κB p65 (Beyotime, AN365, 1 : 1000), NF-κB p50 (Abcam, ab32360, 1 : 1000), p-ERK (Beyotime, AF1891, 1 : 1000), T-ERK (Beyotime, AF1051, 1 : 1000), and TGF-β (Beyotime, AF0297, 1 : 500) were measured in CT26 tumor tissue and CT26 and HCT15 cell lines. Protein concentrations were determined using a BCA kit (Beyotime, China). Protein samples were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). Immunoblotting was performed overnight at 4°C. The membranes were then washed three times with TBST and incubated with the corresponding secondary antibodies (1 : 5000) at room temperature for 2 h. Following incubation, the membranes were washed three times with TBST, and the proteins were visualized using an enhanced chemiluminescence assay kit (Beyotime, China). Images were captured using the G: BOX ChemCR system (Syngene, UK).
5
Protein Isolation and Western Blot Analysis
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Total protein was isolated from ESCC tissues and cells using RIPA buffer (Beyotime). The proteins were separated through sodium dodecyl sulfonate‐polyacrylamide gel (SDS‐PAGE; Solarbio) after being quantified via a BCA Protein Quantification Kit (Vazyme). Then, the samples were transferred onto polyvinylidene difluoride membranes (PVDF; Pall Corporation, New York, NYC, USA) and blocked in skim milk for 2 h. Next, the membranes were incubated with primary antibodies against P‐glycoprotein (P‐gp; ab3366; Abcam), glutathione S‐transferase π (GST‐π; ab53942; Abcam), LASP1 (ab156872; Abcam), or GAPDH (ab8245; Abcam) and corresponding secondary antibody (ab150077; Abcam). The protein bands were analyzed with an enhanced chemiluminescence assay kit (Beyotime).
6
Western Blot Analysis of Protein Markers
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Total protein was extracted using RIPA lysis buffer. Equal amounts of protein (30 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene di uoride membranes (IPVH00010; Millipore Co., Boston, MA, USA). Subsequently, the membranes were incubated with primary antibodies against caspase-3 (ab214430, Abcam; 1:1000), LC3 (ab48394, Abcam; 1:1000), p62 (ab109012, Abcam; 1:1000), H + /K + -ATPase α subunit (D031-3, MBL International Corp.; 1:1000), and H + /K + -ATPase β subunit (D032-3, MBL International Corp.; 1:1000) at 4°C overnight. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab181602, Abcam) served as a loading control. After incubating with the appropriate secondary antibodies for 1 h, the signal was developed using an enhanced chemiluminescence assay kit (Beyotime Biological Technology) and visualized using the ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).
7
Protein Quantification and Western Blot Analysis
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Tumor tissues were lysed in lysis buffer and then centrifuged at 15,000 rpm for 15 min at 4°C. Protein concentration was determined using the BCA kit (Beyotime). A total of 50 µg of protein was subjected to 8–10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, USA). The membranes were blocked for 1 hour at 26°C with 5% bovine serum albumin containing 0.1% Tween-20 and incubated with the primary antibodies (EGF, VEGF, and TGF-β) (Beyotime) (diluted by 1 : 1000) overnight at 4°C. Then, the membranes were washed with TBST three times and incubated with the corresponding secondary antibody (diluted by 1 : 5000) at 37°C for 2 hours. The membranes were then washed again, and the proteins were visualized using an enhanced chemiluminescence assay kit (Beyotime). Images were captured by JY-Clear ECL (JUNYI, China).
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