Briefly, cells were lysed with RIPA lysis buffer supplemented with cocktail (NCM Biotech, Suzhou, China) on a shaking bed at 4 °C for 20 min and centrifuged at 13,000× g at 4 °C for 30 min. Then, the protein supernatants were boiled with SDS loading buffer, separated by 7.5% (wt/vol) SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% milk for 1 h and incubated with the primary antibodies at 4 °C overnight. Finally, the band signals were visualized by an Odyssey Infrared Imagining System (LI-COR, USA) after incubation with the appropriate fluorescent secondary antibodies (CST). The following primary antibodies were used in this study: anti-PD-L1 (CST, 13684 S), anti-EGR1 (CST, 4153 S), anti-VEGFA (Abcam, ab46154), anti-HDAC2 (CST, 5113 S), anti-STAT3 (Abcam, ab226942), anti-phospho-STAT3 (Tyr705) (CST, 9145 S), anti-β-actin (ABclonal, AC026), and anti-H3 (Proteintech, 17168-1-AP).
Odyssey infrared imagining system
Manufactured by LI COR
Sourced in United States
The Odyssey Infrared Imaging System is a versatile and sensitive imaging platform that enables detection and quantification of fluorescently labeled proteins and nucleic acids in a variety of life science applications. The system utilizes near-infrared fluorescence detection to provide high sensitivity and low background signal. It is capable of imaging a range of sample types, including gels, blots, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent tag, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Biosharp (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Skp2 p45 antibody, by Santa Cruz Biotechnology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Phospho eif2α ser51, by Cell Signaling Technology (1 mentions)
Alexa fluor 680, by LI COR (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Irdye 800, by LI COR (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti h3k9me3, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Anti tcea1, by Abcam (1 mentions)
Gapdh, by Merck Group (1 mentions)
Pten antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
β actin antibody, by Merck Group (1 mentions)
7 protocols using odyssey infrared imagining system
1
Protein Expression Analysis by Western Blot
2
Immunoblotting Analysis of SKP2, p27, and HA Proteins
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Cells were harvested at the indicated times and rinsed twice with phosphate-buffered saline (PBS). Cell extracts were prepared with RIPA lysis buffer (Biosharp, Hefei, China, BL504A) and centrifuged at 13,000 g for 30 mins at 4°C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% (w/v) polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 1 hr at room temperature, the membranes were incubated with an appropriate amount of primary antibody at 4°C overnight. The membranes were then incubated with secondary antibodies conjugated to a fluorescent tag (Invitrogen). The band signals were visualized using the Odyssey Infrared Imagining System (LI-COR, USA). Quantification of the band was performed by Image J (National Institutes of Health, US). GAPDH was used as a control. The following antibodies were used: SKP2 p45 Antibody (Santa Cruz, USA, sc-7164, dilution 1:500), GAPDH Antibody (CST, USA, #5174, dilution 1:1000), p27 Kip1 Antibody (CST, USA, #3686, dilution 1:1000), HA-Tag Antibody (CST, USA, #3724, dilution 1:1000).
3
Western Blot Analysis of Protein Signaling
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Protein concentrations of supernatant aliquots were determined by the BCA assay, and 50 µg protein per lane was added to NuPAGE LDS Sample Buffer supplemented with SDS and DTT (Invitrogen). Proteins were resolved using a NuPAGE 12% Bis-Tris Gel and NuPAGE MOPS SDS Running Buffer as described by the manufacturer. Proteins were transferred to Immobilon-P membranes, and membranes were incubated at room temperature with blocking buffer for near infrared fluorescent westerns (Rockland or Odyssey blocking buffer). The membranes were then incubated with antibody [caspase 3, cleaved caspase 3, 4EBP1, phospho-4EBP1 (Thr37/46), eIF4E, eIF2α, phospho-eIF2α (Ser51), or β-actin; 1:1000 dilutions; all from Cell Signaling] at 4 °C overnight. Membranes were then washed 3X in TTBS (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 0.1% v/v Tween 20) and exposed to secondary antibodies conjugated to AlexaFluor 680 or IRDye 800 (1:20,000, Li-COR Biosciences). Membranes were again washed 3X with TTBS, rinsed in PBS (pH 7.4), and the proteins were visualized and quantified using an Odyssey infrared imagining system (Li-COR Biosciences) and Odyssey software.
4
Western Blot Analysis of Cellular Proteins
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Cells were harvested at the indicated times and rinsed twice with PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13,000 g for 30 min at 4 °C. Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5 % (wt/vol) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking with 5 % BSA for 1 h at room temperature, the membrane was incubated with 2.5 μg/mL antibody in 5 % BSA overnight at 4 °C. The membrane was then incubated with a secondary antibody conjugated to a fluorescent tag (Invitrogen). The band signals were visualized and quantified using the Odyssey Infrared Imagining System (LI-COR, USA). The following antibodies were used: Anti-TESC (Abcam, USA), Anti-H3K9me3 (Abcam, USA), and β-actin (Sigma-Aldrich).
5
Western Blot Analysis of Protein Samples
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Cell and tissue extracts were prepared with RIPA lysis buffer (Biosharp, Hefei, China, BL504A) and centrifuged at 13,000×g for 30 min at 4 °C. Then, protein samples were separated by 7.5% (wt/vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). After blocking with 5% milk for 1 h at room temperature, the membranes were incubated with primary antibody at 4 °C overnight. The membranes were then incubated with secondary antibodies conjugated to a fluorescent tag (Invitrogen). The band signals were visualized using an Odyssey Infrared Imagining System (LI-COR, USA).
6
Western Blot Analysis of GALNT8 and TCEA1
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Cells were harvested at the indicated times and rinsed twice with PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13 000 g for 30 min at 4°C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% (w/v) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking with 5% BSA for 1 h at room temperature, the membrane was incubated with 2.5 μg/ml antibody in 5% BSA overnight at 4°C. The membrane was then incubated with a secondary antibody conjugated to a fluorescent tag (Invitrogen). The band signals were visualized and quantified using the Odyssey Infrared Imagining System (LI-COR, USA). The following antibodies were used in this study: Anti-GALNT8 (Abcam, USA), Anti-TCEA1(Abcam, USA) and GAPDH (Sigma-Aldrich).
7
Western Blot Analysis of PTEN Protein
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Cells were rinsed twice with PBS and incubated with RIPA lysis buffer (Millipore, Billerica, MA, USA) on ice for 30 min. Cell extracts were harvested and centrifuged at 13,000 g for 30 min at 4 °C. Then, the lysates were quantified and denatured. Protein samples (10 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 10% (wt/vol) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking, the membrane was incubated with 0.15 μg/ml PTEN antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C or with a β-actin antibody for 1 h (Sigma-Aldrich, St. Louis, MO, USA). The membrane was then incubated with a secondary antibody conjugated to a fluorescent tag (Invitrogen. Carlsbad, CA, USA) for 1 h. The band signals were visualized by the Odyssey Infrared Imagining System (LI-COR) at an 800 channel wavelength.
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