Anti-ICAM-1, anti-eNOS, and anti-COX-2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antiphospho-eNOS, antiphospho-Akt, and anti-Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Western blot analysis of whole cell lysates (30 μg) was performed using the appropriate primary and secondary antibodies. Blots were imaged using a chemiluminescence assay kit from Pharmacia-Amersham (Freiburg, Germany), and band densities were quantified using a Gel Doc 2000 ChemiDoc system and Quantity One software from Bio-Rad (Hercules, CA, USA). Values were normalized to a β-actin loading control.
Gel doc 2000 chemi doc system
Manufactured by Bio-Rad
Sourced in United States, Germany
The Gel Doc 2000 Chemi Doc system is a compact, multipurpose imaging system designed for visualization and documentation of electrophoretic gels and blots. The system features a high-resolution camera, multiple illumination sources, and intuitive software for image capture and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (5 mentions)
Supersignal west pico, by Thermo Fisher Scientific (3 mentions)
Anti icam 1, by Santa Cruz Biotechnology (2 mentions)
Anti phospho akt, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti phospho enos antibody, by Cell Signaling Technology (2 mentions)
Primary and peroxidase conjugated secondary antibodies, by Santa Cruz Biotechnology (2 mentions)
Anti crif1, by Santa Cruz Biotechnology (2 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Anti enos, by Santa Cruz Biotechnology (1 mentions)
Anti cox 2, by Santa Cruz Biotechnology (1 mentions)
Anti phospho enos, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti akt antibody, by Cell Signaling Technology (1 mentions)
Total akt, by Santa Cruz Biotechnology (1 mentions)
Anti nos3, by Santa Cruz Biotechnology (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Anti total creb, by Santa Cruz Biotechnology (1 mentions)
Anti phospho creb, by Santa Cruz Biotechnology (1 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
8 protocols using gel doc 2000 chemi doc system
1
Western Blot Analysis of eNOS and Akt
2
Quantifying Phospho-eNOS Levels in Rat Aorta
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Anti-phospho-eNOS antibody was purchased from Cell Signaling (Beverly, MA, USA). Anti-NOS3, anti-β-actin, anti-phospho-Akt and total Akt antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed by boiling 30 µg of whole cell lysate or 30 µg of tissue homogenate (obtained from rat aorta) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer, before separation by electrophoresis and transfer to a nitrocellulose membrane. After incubation in appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), chemiluminescent signaling was developed using Super Signal West Pico or Femto Substrate from Thermo Fisher Scientific (Pierce, Rockford, IL, USA). Blots were imaged and band densities quantified with a Gel Doc 2000 Chemi Doc system using Quantity One software from Bio-Rad (Hercules, CA, USA). Values were normalized to a β-actin loading control.
3
Immunoblotting and Immunoprecipitation Assays
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Immunoblotting was performed as described previously [41 (link), 44 (link)]. Briefly, 50 μg of protein lysates from untreated cells and cells exposed to Triptolide were boiled in SDS-PAGE gel loading buffer (Bio-Rad), subjected to SDS-PAGE, transferred to nitrocellulose filter, and probed with the specified primary antibody and the appropriate peroxidase-conjugated secondary antibody (Santa Cruz Biotech). Chemiluminescent signal was developed using Super Signal West Femto substrate (Pierce), blots imaged with a Gel Doc 2000 Chemi Doc system (BioRad). For immunoprecipitation studies, cells were lysed in IP buffer [50mM Tris (pH 7.4), 150 mM NaCl, 5% Glycerol, 1% Triton-X100) containing Complete EDTA-free protease inhibitor mixture (Roche). Cell lysates were incubated with indicated antibodies for overnight at 4°C. Next day, immune complex was captured using Dynabeads Protein G (Invitrogen, USA) by incubating for additional 45 minutes. Immune complexes were washes four times, eluted with SDS-PAGE gel loading buffer and were separated by SDS-PAGE as described above. For endogenous immunoprecipitation of P53 and sirt3, cells were cross-linked with formaldehyde (1%) for 15 minutes to stabilize the complex before harvesting cells in IP buffer.
4
Western Blot Analysis of Signaling Proteins
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Anti-CRIF1, anti-phospho Akt, anti-phospho CREB and anti-total CREB antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti RhoGDI2 antibody was purchased from Spring Bioscience (Pleasanton, California, USA). Anti-β-actin and anti-total Akt antibodies were from Cell Signaling Technology (Beverly, MA, USA). Western blot analysis was performed by adding 30 μg of cell lysate to sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer followed by boiling and separation by electrophoresis and transfer onto nitrocellulose membranes. After incubation with appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate (Thermo Fisher Scientific, Pierce, Rockford, IL). Blots were imaged and band densities quantified with a Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA). Values were normalized relative to β-actin as the loading control. Recombinant ADM2 (Intermedin-53, human) was purchased from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA).
5
Western Blot Analysis of Cell Signaling
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Anti-VCAM-1, anti-ICAM-1, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-shcA antibodies were purchased from Becton Dickinson (Fullerton, CA, USA). Anti-phospho-p38 and anti-p38 antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Whole cell lysates (50 µg) were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel loading buffer, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Chemiluminescent signaling was developed using Super Signal West Pico or Femto substrate (Pierce, Rockford, IL, USA). The blots were imaged and band densities were quantified with a Gel Doc 2000 Chemi Doc system and Quantity One software (Bio-Rad, Hercules, CA, USA). Values were normalized to those of β-actin as the loading control.
6
Quantifying eNOS Protein Expression
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Anti-phospho-eNOS antibody was purchased from Cell Signaling (Beverly, MA, USA). Anti-NOS3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blotting analysis was performed by adding 30 μg tissue homogenate (obtained from rat aorta) to SDS–PAGE loading buffer followed by boiling and separation by electrophoresis and transfer onto nitrocellulose membranes. After incubation with appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate from Thermo Fisher Scientific (Rockford, IL, USA). Blots were imaged and band densities quantified with a Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA, USA). Values were normalized relative to β-actin loading control.
7
Western Blot Analysis of TGF-β Signaling
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Mouse monoclonal anti-β-ACTIN, rabbit polyclonal anti-phospho Smad2, rabbit monoclonal anti-Smad2, rabbit monoclonal anti-phospho Smad3, rabbit polyclonal anti-Smad3, rabbit polyclonal anti-TGF-β, rabbit monoclonal anti-PCNA, and rabbit monoclonal anti-SMURF2 were obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal anti-CRIF1 was obtained from Abcam (Cambridge, UK). Rabbit polyclonal anti-SMAD7 antibody was obtained from Invitrogen (Carlsbad, CA, USA). For Western blot, 15 μg of whole cell lysates was loaded and separated on 6–12% SDS-PAGE gels by electrophoresis, followed by incubation in the appropriate primary and secondary antibodies. For each western blot quantified, the experiment was repeated for a minimum of three times. Blots were imaged using a chemiluminescence assay kit (Miracle-Star Western Blot Detection System; Intron Biotechnology, Seongnam, Republic of Korea) and EZ-Western Lumi Femto (Daeil Lab Service, Seoul, Republic of Korea), and band densities were quantified on a Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA, USA). Values were normalized to β-actin (loading control).
8
Immunoblotting of Cellular Proteins
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Rabbit polyclonal Anti-Crif1, rabbit polyclonal anti-vascular cell adhesion molecule-1 (VCAM-1) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Mouse monoclonal C/EBP homology protein (CHOP), rabbit monoclonal eukaryotic initiation factor 2 (eIF2)-α antibodies were purchased from Cell signaling (USA). Mouse monoclonal antibodies against OXPHOS complex subunits (NDUFA9, SDHA, UQCRC2, COX4 and ATP5A1) were purchased from Invitrogen, mouse polyclonal anti-phospho-ser36-p66shc antibody from Calbiochem (CA, USA) and rabbit polyclonal anti-Shc antibody from BD Biosciences (NJ, USA). Western blotting of 30 µg whole-cell lysates was performed using appropriate primary and secondary antibodies. Blots were imaged using a chemiluminescence assay kit from Pharmacia-Amersham (Freiburg, Germany), and band densities were quantified using a Gel Doc 2000 Chemi Doc system and the Quantity One software from Bio-Rad (Hercules, CA). Values were normalized to a β-actin loading control.
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