After recovery of the frozen tissue samples, protein was extracted using the natural membrane protein extraction kit ProteoExtract (M-PEK) (MERCK Co., Kenilworth, NJ, USA), and the protein concentration was determined using a Bio-Rad protein assay kit (Hercules, CA, USA) according to manufacturer’s instructions. Subsequently, 40 μg of protein was loaded on an 8% SDS–polyacrylamide gel (stacking gel 100 V, resolving gel 200 V) for 1 h at an electrode constant current of 300 mA. The proteins in the gel were transferred onto a nitrocellulose membrane (Kexing Co., Beijing, China), blocked for 1 h at 37 °C in 5% skim milk powder, and incubated overnight at 4 °C with sheep anti-rat TGFβ1 (ab208466; Abcam, Cambridge, UK) and Smad7 (66,478; Proteintech, Wuhan,China) antibodies [25 (link)]. Horseradish peroxidase-labeled rabbit anti-sheep secondary antibody was added (1:400; Nakasugi Jinqiao Co., Beijing, China) and incubated at 37 °C for 1 h. The signal was detected using the enhanced chemiluminescence reagent (Abcam). Positive bands were analyzed using Gel-Pro 4.0 gel optical density analysis software, and the cumulative optical density (IOD value) was measured, with the R value representing the relative protein content as follows: R = reference IOD value of PLSCR (target)/reference IOD value of GAPDH (reference protein).
Enhanced chemiluminescence reagent
Manufactured by Abcam
Sourced in United States, China
Enhanced chemiluminescence reagent is a laboratory reagent used to detect and quantify proteins in western blot analysis. It produces a luminescent signal in the presence of the target protein, which can be measured using a luminometer or X-ray film.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (3 mentions)
Ab9485, by Abcam (2 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab55878, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Ripa lysis buffer, by Teknova (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab6728, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Beyotime (1 mentions)
Skim milk, by Beyotime (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
Ab209847, by Abcam (1 mentions)
Ab198921, by Abcam (1 mentions)
Ab115730, by Abcam (1 mentions)
Anti mmp9, by Abcam (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti proliferating cell nuclear antigen, by Abcam (1 mentions)
Ripa lysis buffer, by Elabscience (1 mentions)
12 protocols using enhanced chemiluminescence reagent
1
Membrane Protein Extraction and Western Blot Analysis
2
Autophagy Protein Expression Analysis
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Western blot analysis was performed to measure the expression of beclin-1 and LC3-II/I, proteins associated with autophagy (25 (link)). HK-2 cells were lysed using RIPA lysis buffer (Teknova, Inc.) at 4°C for 10–15 min. Protein concentration was determined using bicinchoninic acid assay method. Equal amounts of 50 µg protein extract were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk diluted in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 90 min. The membranes were then immunoblotted for Beclin1 (1:1,000; cat. no. ab55878; Abcam), LC3 (1:1,000; cat. no. ab48394; Abcam), and β-actin (1:1,000; cat. no. ab3280; Abcam) at 20–27°C for 2 h before incubation with secondary antibodies conjugated to horseradish peroxidase-conjugated goat anti-rabbit Immunoglobulin G (1:5,000, cat. no. ab7074; Abcam) at 37°C for 20 min and visualized with enhanced chemiluminescence reagent (Vazyme). Immunoreactive bands were analyzed using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).
3
Protein Expression Analysis by Western Blot
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Cells were lysed using RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.25% deoxycholic acid disodium salt, 1% NP40, 150 mM NaCl and 0.1% SDS). After sonication, cells were centrifuged at 12,000 × g for 15 min at 4°C. Protein concentration was determined using a BCA assay. Then, 30 µg of cell extracts were subjected to 10–12% SDS-PAGE electrophoresis and transferred to PVDF membranes. Following protein transfer, the membranes were blocked with 5% milk and then incubated with specific primary antibodies (1:1,000) at 4°C overnight followed by a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. nos. ab6721 and ab6728; Abcam) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence reagent (cat. no. 32106; Thermo Fisher Scientific, Inc.).
4
Protein Extraction and Western Blot Analysis
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After the extraction of proteins from serums and cells via radioimmunoprecipitation assay (RIPA) lysis solution (Beyotime, Shanghai, China), 60 μg quantified proteins were isolated on 10% sodium dodecyl sulfate–polyacrylamide gel for 2 h, followed by the transferring of proteins onto the polyvinylidene fluoride membranes (Beyotime). Then, 5% skim milk (Beyotime) was applied for blocking membranes for 3 h, which were incubated with primary antibodies from Abcam (Cambridge, United Kingdom): anti-Hexokinase 2 (anti-HK2; ab209847, 1:1000), anti-Glucose Transporter 1 (anti-GLUT1; ab115730, 1:1000), anti-SOX13 (ab198921, 1:1000) and anti-GAPDH (ab9485, 1:3000) overnight at 4 °C Following the incubation of secondary antibody (Abcam, ab205718, 1:5000) for 1 h, the immunoreactive signals were assayed through enhanced chemiluminescence reagent (Abcam), and the gray levels were analyzed via the Image J software (NIH, Bethesda, MD, USA).
5
Quantitative Protein Analysis of MDA-MB-231 Cells
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Total protein was extracted from 1×106 MDA-MB-231 cells using RIPA lysis buffer (Elabscience, cat. No. E-BC-R327) and quantified using a Pierce™ BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.). Following denaturation, electrophoresis of 30 µg protein/lane was performed using 12% SDS-PAGE. Following gel transfer onto PVDF membranes, the membranes were blocked in 5% fat-free milk for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-MMP2 (1:1,000; cat. no. ab215986; Abcam), anti-MMP9 (1:1,000; cat. no. ab219372; Abcam), anti-GAPDH (1:1,000; cat. no. ab181602; Abcam), anti-Ki67 (1:1,000; cat. no. ab92742; Abcam), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. ab92552; Abcam), anti-CDCA5 (1:1,000; cat. no. ab192237; Abcam) and anti-PDS5A (1:500; cat. no. 203627-T34; Sino Biological Inc.). Next, the membranes were incubated with a goat anti-rabbit HRP-conjugated IgG secondary antibody (1:2,000; cat. no. ab6721; Abcam) at room temperature for 4 h. The protein bands were visualized using an enhanced chemiluminescence reagent (cat. no. abs920, Absin). The protein expression levels were semi-quantified using ImageJ software (v1.46; National Institutes of Health) with GAPDH as the loading control.
6
Protein Extraction and Western Blot Analysis
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Protein was extracted using a natural membrane protein extraction kit ProteoExtract (Chemical Book Co., Beijing, China), and the protein concentration was determined using a Bio-Rad protein assay kit (Noble-Ryder Co., Beijing, China). Subsequently, 40 μg of protein was loaded on an 8% SDS–polyacrylamide gel (stacking gel 100 V, resolving gel 200 V) for 1 h at a constant current of 300 mA. Proteins in the gel were transferred onto a nitrocellulose membrane (Kexing Co., Shenzhen, China) and blocked with 5% skim milk for 1 h at 37 °C and incubated overnight at 4 °C with sheep anti-rat PLSCR monoclonal antibody (1:200, PAB781Hu01; Abcam, Guangzhou, China). Horseradish peroxidase-labeled rabbit anti-sheep secondary antibody was then added (1:400) and the mixture was incubated at 37 °C for 1 h. After washing the membrane, the signal was detected with enhanced chemiluminescence reagent (Abcam, Guangzhou, China). Positive bands were analyzed using Gel-Pro 4.0 gel optical density analysis software, and the IOD value was measured, with the R value representing the relative protein content as described above.
7
Western Blot Analysis of Apoptosis-Related Proteins
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Following the obtaining of proteins by Radio-Immunoprecipitation Assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA), 40 μg proteins were split on 10% sodium dodecyl sulfate–polyacrylamide gel (Invitrogen) for 2 h. 5% skim milk (Thermo Fisher Scientific) was implemented to block the non-specific binding signals after the transferring of proteins onto the polyvinylidene fluoride membranes (Thermo Fisher Scientific). Then membranes were incubated with primary antibodies: anti-CREBRF (Abcam, Cambridge, UK, ab26262, 1:1000), anti-poly-ADP-ribose polymerase (anti-PARP; Abcam, ab74290, 1:1000), anti-Cleaved PARP (Abcam, ab30264, 1:1000), anti-Cleaved caspase-3 (Abcam, ab2302, 1:1000) and anti-β-actin (Abcam, ab8227, 1:3000) for 3 h at room temperature. Subsequently, the secondary antibody (Abcam, ab205718, 1:5000) was exploited to bind to primary antibodies. 1 h later, the detection of combined signals was administered by the enhanced chemiluminescence reagent (Abcam). Ultimately, ImageLab software version 4.1 (Bio-Rad Laboratories, Hercules, CA, USA) was used for image acquisition and densitometric analysis previously [22 (link)].
8
Protein Extraction and Western Blot Analysis
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For extraction of protein, RIPA lysis buffer (Keygen) was used to lyse cells or tissues. After measuring the protein concentration using a BCA protein assay kit (Abcam), electrophoresis was performed with SDS‐PAGE to separate the protein. Next, the protein was transferred onto PVDF membranes (Invitrogen), followed by blocking in nonfat milk (5%; Beyotime). After that, the membranes were probed with primary antibodies (overnight, 4°C). The primary antibodies containing Cyclin D1 (1:1000, ab226977), MMP9 (1:2000, ab76003), PDL1 (1:1000, ab205921) and GAPDH (1:2000, ab9485) were all provided by Abcam. After incubation with secondary antibody (1:3000, ab205718, Abcam), enhanced chemiluminescence reagent (Abcam) was used to visualize the blot signal.
9
Protein Extraction and Western Blot Analysis
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A Whole Cell Extraction Kit (cat. no. OP-0003; Epigentek Group, Inc.) was used to extract total protein from the osteosarcoma cells. Protein concentration was determined using a BCA Protein Quantification Kit (cat. no. E112-01; Vazyme Biotech Co., Ltd.). Subsequently, 45 µg proteins were separated by 10% SDS-PAGE and were transferred onto PVDF membranes. Membranes were blocked using 5% skimmed milk at room temperature for 1 h and incubated with the primary antibodies (diluted with 5% skimmed milk) against E-cadherin (cat. no. ab15148; 1:1,000; Abcam), N-cadherin (cat. no. ab18203; 1:1,000; Abcam), and matrix metalloproteinase-9 (MMP-9) antibody (cat. no. ab38898; 1:2,000; Abcam) and β-actin (cat. no. sc-69879; 1:3,000; Santacruze) overnight at 4°C Membranes were then incubated with goat anti-mouse IgG H&L (HRP) (cat. no. ab6789; 1:2,000; Abcam) or goat anti-rabbit IgG H&L (HRP) (cat. no. ab6721; 1:2,000; Abcam) at room temperature for 2 h. Enhanced chemiluminescence reagent (cat. no. WBKLS0100; Beijing Xinjingke Biotechnologies Co., Ltd.) was used to detect the signal on the membrane. ImageJ software (v1.8.0; NIH) was used to quantify the gray value of protein bands.
10
Immunoprecipitation and Western Blot Analysis
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