Cells (2 × 106) were taken from each group, added to a celllysis buffer (100 µL) until full cleavage was achieved, and boiled (100 °C) for 5 min. The insoluble material was removed by centrifugation (5000×g for 5 min). The proteins in each sample (50 µL) were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were sequentially blocked with 5 % nonfat milk in PBS for 2 h and 5 % bovine serum albumin in PBS at 4 °C overnight, and then incubated with primary antibodies for 90 min at room temperature. The primary antibodies used were rabbit polyclonal immunoglobulin (IgG) antibodies against occludin (Ab31721; Abcam, UK; 1:250 dilution), claudin-1 (Ab15098; Abcam, UK; 1:25 dilution), and ZO-1 (Sc-10804; Santa Cruz Biotechnology, USA; 1:100 dilution). Peroxidase-conjugated goat anti-rabbit IgG (A0208; Beyotime, China; 1:1000 dilution) and chemiluminescence fluids A and B (1:1) were incubated with the membranes for 1 h and 1 min, respectively. The reactive bands were detected by chemiluminescent reagents (170-5061; Bio-Rad, USA). A Tanon-5200imager (Tanon, China) was employed for image acquisition, and Image-pro plus 6.0 software (MediaCybernetics, USA) was used to analyze the gray values of the bands. All the grey values of the bands were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Chemiluminescent reagent
Manufactured by Bio-Rad
Sourced in United States
Chemiluminescent reagent is a laboratory product that emits light when mixed with a target analyte. It is used to detect and quantify the presence of specific biomolecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody, by Proteintech (2 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab15098, by Abcam (1 mentions)
Ab31721, by Abcam (1 mentions)
Sc 10804, by Santa Cruz Biotechnology (1 mentions)
A0208, by Beyotime (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Eba 1052, by Elpis Biotech (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Imagequant las 4000 series, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Lc3 polyclonal antibody, by Proteintech (1 mentions)
13 protocols using chemiluminescent reagent
1
Immunoblotting Analysis of Tight Junction Proteins
2
Protein Extraction and Western Blot Analysis
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SH-SY5Y cells and mouse brain tissue samples were lysed in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Gyeonggi-do, Korea), and centrifuged for 30 min at 4 °C in 15,000 rpm. The supernatant was boiled with sample buffer (EBA-1052, Elpis Biotech., Daejeon, Korea) for 5 min. The samples were loaded by 12% SDS-PAGE and were then transferred to a nitrocellulose membrane. The membranes were incubated with 5% skim milk in TBST for 1 h and were incubated with primary antibody overnight at 4 °C. After that these were incubated with HRP-conjugated antibody for 2 h. The protein bands were utilized chemiluminescent reagents (Amersham, Franklin Lakes, NJ, USA), and detected using Chemidac system (BioRad, USA).
3
Western Blot Quantification Protocol
4
Protein Quantification and Immunoblotting Protocol
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Cell lines extracts were generated and subjected to protein quantification by BCA (Thermo Scientific). Protein extracts were subjected to electrophoresis on sodium dodecyl sulphate–polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membrane (GE Healthcare). Proteins were detected with the relevant antisera using either chemiluminescent reagents (Bio-Rad) or by Odyssey (LI-COR).
Cap m7GTP Sepharose (GE Healthcare) column pull-down was performed on 400 µg of total extracts according to manufacturer instructions. Densitometry was performed with Fiji (Fiji Is Just ImageJ) software according to standard procedure.
Cap m7GTP Sepharose (GE Healthcare) column pull-down was performed on 400 µg of total extracts according to manufacturer instructions. Densitometry was performed with Fiji (Fiji Is Just ImageJ) software according to standard procedure.
5
Profiling TNBC Signaling Pathways
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TNBC cell lines were maintained in serum-free media overnight (control) or treated with CF33 over 24 hr with cell harvests at 1-, 4-, 8-, and 24-hr intervals. Whole-cell extracts were fractionated by SDS-PAGE and transferred to membrane using specified manufacturer’s instruction for the apparatus (Bio-Rad, Hercules, CA). Membrane was incubated in 5% nonfat milk for 2 hr and then incubated with antibodies against p-Akt (Ser473), total-Akt, phospho-ERK (p-ERK; Thr 202/Tyr 204), total-ERK, and gapdh (all 1:1,000) overnight at 4°C (antibodies from Cell Signaling Technologies). After washing, membranes were developed with chemiluminescent reagents according to manufacturer’s protocol (Bio-Rad).
6
Western Blot Analysis of Protein Expression
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Total protein was extracted using RIPA buffer and quantified using Bradford’s’ reagent. Protein was loaded onto 10% SDS-PAGE, electrophoresed at 110 V for 2 hours and subsequently transferred onto PVDF membranes (BIO-RAD, USA). The membranes were incubated with mAb #12857, and mAb #4970 (Cell Signalling Technologies, USA) at a dilution of 1:1000 followed by incubation with mAb #7074 (Cell Signalling Technologies, USA) at a dilution of 1:10000. The blot was exposed to chemiluminescent reagent (BIO-RAD, USA) for visualization.
7
SARS-CoV-2 Spike Protein Detection
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Briefly, purified pseudovirion virus pellets of SFL, Sdel18, SG614, SFko, or VSV-G were passed through a 20% sucrose layer and cell lysates were resuspended in 30 μl RIPA buffer, and incubated on ice for 30 min with an interval vortex. Then the samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membranes. After blocking with 5% milk, the membranes were blotted with SARS-CoV-2 (2019-nCoV) Spike Antibody, Rabbit PAb (1:2000) (Sinobiological Inc., China) and incubated in blocking solution overnight at 4℃, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:3000) (Proteintech, China) and visualization with Chemiluminescent Reagent (Bio-Rad, USA).
8
Protein Expression and Immunoblotting Analysis
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293T cells were transfected with indicated plasmids as mentioned above and harvested after 24 h by addition of lysis buffer, and incubated on ice for 15 min. Cell lysates were centrifuged at 12,000 ×g for 15 min before the supernatants were either subjected to immunoprecipitation (IP) or denatured directly at 100°C for 10 min. Denatured cell lysates were separated by SDS-PAGE and transferred to PVDF membranes using 200 mA for 70 min. For immunoblotting, indicated primary antibodies were used to incubate the membranes for 3 h at RT or overnight at 4°C; HRP-conjugated goat anti-mouse or goat anti-rabbit IgG were used as secondary antibodies. The bands were visualized with chemiluminescent reagent (#1705061, Bio-Rad, Hercules, CA, USA) and were imaged by an ECL imaging system (LI-COR biosciences), and amounts of β-actin, GAPDH or TPB of each sample were used as controls to demonstrate equal loading of protein samples among lanes.
9
Western Blot Analysis of LC3 and GAPDH
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Briefly, Cells treated with different antibodies were collected and lysed in RIPA buffer (beyotime), containing protease inhibitor cocktail (Abcam), and incubated on ice for 1 h with an interval vortex. The lysates underwent centrifugation at 12,000 rpm for 15 min at 4 °C. The total protein concentrations in the resulting supernatant fractions were determined using a BCA protein assay kit (beyotime). Then the equal amounts of protein samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membranes. After blocking with 5% milk, the membranes were blotted with LC3 polyclonal antibody (Proteintech), Rabbit PAb (1:2000) and GAPDH antibody (Servicebio), Rabbit PAb (1:3000) incubated in blocking solution overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:3000) (Proteintech) and visualization with Chemiluminescent Reagent (Bio-Rad, USA).
10
Hepatic Protein Quantification Protocol
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Total liver protein lysates were separates on 10% and 12% SDS–polyacrylamide gel and transferred on nitrocellulose membrane. Membranes were blocked with 5% BSA in TBS–0.01% Tween 20 and probed with specific antibodies against PGC-1β (ab176328) and GCL-c (ab53179) purchased from Abcam. Nuclear encoded β-actin (Abcam) were used as loading control. Membranes were finally incubated with HRP conjugated secondary antibodies and developed with a chemiluminescent reagent (Biorad, California, USA).
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