A549 cancer cells were infected with GLV-1h312, GLV-1h460 and GLV-1h462 respectively, at an MOI of 0.1 in the presence or absence of doxycycline (1 µg/ml). After one day, the medium was aspirated and the cell layer lysed in SDS sample buffer, transferred to fresh 1.5 ml tube and heated to 99°C for 10 min. Protein lysates were separated by 10% SDS-Polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Whatman GmbH, Dassel, Germany). After blocking, the membrane was incubated with anti-human-tyrosinase goat polyclonal antibody (sc-7833, Santa Cruz Biotechnology Inc, Santa Cruz, USA), anti-beta-glucuronidase rabbit polyclonal antibody (G5420, Sigma-Aldrich), anti-beta-actin mouse monoclonal antibody (ab6276, Abcam, Cambridge, UK), anti-RFP rabbit polyclonal antibody (AB231, Evrogen, Moscow, Russia), anti-GFP rabbit polyclonal antibody (sc-8334, Santa Curz Biotechnology Inc) and anti-TetR mouse monoclonal antibody (Clone 9G9, Clontech, Mountain View, USA) respectively. The protein bound primary antibodies were detected using horseradish peroxidase conjugated rabbit anti-mouse (ab6728, Abcam), goat anti-rabbit (ab6721, Abcam) or rabbit anti-goat (ab6741-1, Abcam) secondary antibodies, followed by enhanced chemiluminescence detection with the molecular imager ChemiDox XRS+ from Bio Rad (Bio Rad, Munich, Germany).
Chemidox xrs
Manufactured by Bio-Rad
Sourced in United States
The ChemiDox XRS is a chemiluminescence detection system designed for imaging and quantifying protein and nucleic acid samples. It features a high-resolution CCD camera, motorized filter wheel, and advanced software for image capture and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (4 mentions)
Facscalibur, by BD (2 mentions)
Facsaria 2 sorp, by BD (2 mentions)
Goldview 2 nuclear staining dye, by Solarbio (2 mentions)
Gapdh, by Proteintech (2 mentions)
Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Ibind western device, by Thermo Fisher Scientific (2 mentions)
Trans blot cell, by Bio-Rad (2 mentions)
α galactose m86 monoclonal igm antibodies, by Enzo Life Sciences (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Hybond n nitrocellulose membrane, by GE Healthcare (2 mentions)
Denhardt s solution, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent nucleic acid detection module kit, by Thermo Fisher Scientific (2 mentions)
Bio dot apparatus, by Bio-Rad (2 mentions)
Ab6741 1, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab6728, by Abcam (1 mentions)
Whatman, by Cytiva (1 mentions)
32 protocols using chemidox xrs
1
Western Blot Analysis of GLV-1h Virus Infection in A549 Cells
2
Protein Characterization by SDS-PAGE
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SDS-PAGE was also used to determine the titers of targets with an Fc or His tag based on the band intensity. Briefly, 10 μL of CM was analyzed under both reducing and non-reducing conditions using a 4–12% Bis-Tris gel and 1x MES running buffer (Invitrogen). Gels were stained with Coomassie blue and imaged using ChemiDox XRS+ (Bio-Rad).
Purified proteins were analyzed on a 4–20% Tris-glycine stain-free gel in the presence of 1x Tris-glycine running buffer (Bio-Rad). Gels were typically imaged using the stain-free gel application protocol in Image Lab (Bio-Rad) before staining with Coomassie blue.
To analyze the glycosylation status of the protein via SDS-PAGE inFig 1D and S3 Fig , 2.5 μg of protein was deglycosylated using either non-reduced or reduced rapid PNGase F (cat. no. P0711S for non-reducing format, P0710S for reducing format, NEB) in a total reaction volume of 10 μL at 50°C for 30 min. Untreated protein samples were subjected to the same protocol but without rapid PNGase F addition. They were subsequently analyzed by SDS-PAGE under non-reducing or reducing conditions. Gels were stained with Coomassie blue or directly visualized with the stain-free imaging.
Purified proteins were analyzed on a 4–20% Tris-glycine stain-free gel in the presence of 1x Tris-glycine running buffer (Bio-Rad). Gels were typically imaged using the stain-free gel application protocol in Image Lab (Bio-Rad) before staining with Coomassie blue.
To analyze the glycosylation status of the protein via SDS-PAGE in
3
Western Blot Analysis of Phospho-CREB
4
Western Blot Analysis of β-Galactosidase
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The E. coli strain MG1655 from Procedure C were harvested and suspended in lysis buffer containing 50 mM sodium phosphate (pH 7.0), 10 mM imidazol, and 300 mM NaCl. The OD was normalized using lysis buffer, after which the crude extract was prepared using sonication on ice 5 s × 5 times. Anti-β-galactosidase antibody (Biotin) ab6645 was purchased from abcam (Cambridge, UK) and used at a 105-fold dilution. Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate (Biorad) was used as the secondary antibody. Bands were detected using the ECL Select Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and were recorded on ChemiDox XRS (Biorad). Band density was quantified using ImageJ software.
5
Investigating Ginseng-based Apoptosis Modulation
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RPMI 1640 Medium and Penicillin-Streptomycin Solution were from Hyclone (USA). FBS was from Tianhang Biological Technology (Hangzhou, China). PBS was from CORNING (NY, USA). 0.25% trypsin and CCK-8 were from Beyotime (Nantong, China). SD (Ginseng, Atractylodes, Poria, Licorice) was from Department of Traditional Chinese medicine of the First Affiliated Hospital of Bengbu Medical College (Bengbu, China). 0.22μm syringe filter was from Millpore (Boston, USA). Hochest 33342 was from Sigma (Saint Louis, USA). Verapamil was from Shanghai Pharmaceutical Group Co., Ltd. (Shanghai, China). AV and PI were from BD (Franklin Lakes, USA). Antibodies against Bax and Bcl-2 were from Abcam (Cambridge, England). Substrate Reagent was from Millpore (Boston, USA). β-actin antibody was from SANTA CRUZ (Dallas, USA). Horseradish peroxidase-labeled goat anti-rabbit IgG was from Biosharp (Hefei, China). Flow cytometers BD FACS ARIA II SORP and BD FACSCalibur: BD (Franklin Lakes, USA). Electrophoresis apparatus and gel imaging system: BioTeK Synergy 2 (BioTeK, USA). Power supply and gel imaging system ChemiDox XRS: Bio-Rad (Hercules, USA).
6
Evaluating mRNA Binding Efficiency of DMP-039 Nanoparticles
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We used different molecular ratios of DMP-039 nanoparticles to evaluate the binding ability of mRNA. When DMP-039 binds with mRNA, the molecular weight increases, the migration in the gel will be blocked, and the bands that appear will be shortened. mBim (0.5 μg) mixed with DMP-039 at different ratios was electrophoresed on a 1% (w/v) agarose gel for 15 min at 120 V. The gel was then stained with GoldView II Nuclear Staining Dye (Solarbio), and the bands were detected with an E-gel imager (Bio-Rad, ChemiDox XRS, USA).
7
Protein Expression and Analysis in E. coli
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E. coli cells were cultivated at 37°C in Luria-Bertani medium (LB medium, 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) supplemented with corresponding antibiotics until their OD600 reached 0.6. Then, cells were cultured at 30°C for 4 h after addition of 1 mM isopropyl ß-D-thiogalactoside (IPTG). Cells were subsequently collected from 1.5 mL of bacterial cultures by centrifugation and re-suspended in sodium dodecyl sulfate (SDS) sample buffer. Proteins were heated in boiling water for 5 min. The resulting supernatant was analyzed by SDS-polyacrylamide gel electrophoresis and Western blot.
For Western blot analysis, protein samples were separated on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking non-specific binding using 5% skim milk for 1 h, the blots were incubated with anti His-tag monoclonal antibody or anti S-tag antibody at 4°C overnight. Membranes were washed three times with TTBS buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) and incubated with an appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. After being washed three times with TTBS, direct chemiluminescence imaging of the blots was performed using the ChemiDox XRS (BioRad) imaging system.
For Western blot analysis, protein samples were separated on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking non-specific binding using 5% skim milk for 1 h, the blots were incubated with anti His-tag monoclonal antibody or anti S-tag antibody at 4°C overnight. Membranes were washed three times with TTBS buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) and incubated with an appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. After being washed three times with TTBS, direct chemiluminescence imaging of the blots was performed using the ChemiDox XRS (BioRad) imaging system.
8
Western Blot Protein Analysis
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Equal amounts of proteins were separated by electrophoresis on a sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (PR05509, Millipore, Cork, Ireland). Antibodies included GOLM1 (1:4000; Proteintech, Cat# 15126-1-AP), LIMK1 (1:1000; affinity, Cat#AF6345), CBX1 (1:1000; Proteintech, Cat#10241-2-AP), and GAPDH (1:5000; Proteintech, Cat# 60004-1-Ig). Secondary antibodies were goat anti-rabbit IgG (1:5000, Abcam, Cat# AB97051) or goat anti-mouse IgG (1:5000, Abcam, Cat# AB97023). ChemiDox XRS (Bio-Rad) was used for detection, and pictures were analyzed with ImageJ software (NIH, Bethesda, MD).
9
Western Blot Analysis of Protein Expression
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Protein extracted from the transfected cells with RIPA lysis buffer (BOSTER, Wuhan, China) was separated by 10% SDS-PAGE and then transferred to nitrocellulose filter membranes. After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated overnight at 4 ℃ with primary antibodies as shown in Table S2 . After incubating with the secondary antibodies, protein bands were visualized by a chemiluminescence system (ChemiDox XRS+, Bio-Rad, CA, USA) and then analyzed using Image Lab software from Bio-Rad.
10
Ferroptosis Marker Protein Analysis
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The neurons were lysed with lysis buffer (Beyotime, Nantong, China), and the proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 5% nonfat milk at room temperature for 1 hour, and thereafter incubated with primary antibodies [anti-GPX4 (1:5000; ab125066, rabbit, Abcam), anti-xCT (1:1000; ab175186, rabbit, Abcam), anti-β-actin (1:5000; sc47778, mouse, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-GAPDH (1:2000; CST2118, rabbit, Cell Signaling Technology, Boston, MA, USA)] overnight. GPX4 and xCT are both key ferroptosis markers. The blots were then incubated with secondary antibodies (horseradish peroxidase-linked anti-mouse IgG (CST7076, 1:3000; Cell Signaling Technology) and horseradish peroxidase-linked anti-rabbit IgG (CST7074, 1:3000; Cell Signaling Technology) at room temperature for 1 hour. Finally, the blots were visualized using an enhanced chemiluminescence system (ChemiDox XRS; Bio-Rad, Hercules, CA, USA). All grayscale values were normalized to that of β-actin or GAPDH using ImageJ software (National Institutes of Health).
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