Total protein was extracted by the peqGOLD TriFast procedure (Peqlab Biotechnology GmbH, Erlangen, Germany). Proteins were separated on 10% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After blocking, the membranes were probed with one of the following antibodies: anti-HIF-1α (BD Biosciences, Franklin Lakes, NJ, USA), anti-HIF-2α (LifeSpan BioSciences, Inc., Seattle, WA, USA), anti-PKM2 (Cell Signaling Technologies, Danvers, MA, USA), anti-PKM2 (Y105; Cell Signaling Technologies), and anti-β-actin (Abcam, Cambridge, United Kingdom). All primary antibodies were diluted 1:1000. Then membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Antibody complexes were visualized by enhanced chemiluminescence using an ECL kit (GE Healthcare, Little Chalfont, United Kingdom). An image reader (FluorchemTM IS-8900, Alpha Innotech, San Leandro, CA, USA) was used to visualize and quantify western blot bands. Expression was quantified using band intensity values (in arbitrary units), which were normalized to that of β-actin.
Fluorchem is 8900
Manufactured by Bio-Techne
Sourced in United States
The Fluorchem IS-8900 is a laboratory imaging system designed for the detection and analysis of fluorescent signals. It features a high-resolution camera, adjustable lighting, and specialized software for image capture and processing.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescent luciferase kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Anti hif 1α, by BD (1 mentions)
Anti pkm2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Abcam (1 mentions)
Western blot luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Electroblotting, by Bio-Rad (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab133303, by Abcam (1 mentions)
Ab4207, by Abcam (1 mentions)
Rpn2232, by GE Healthcare (1 mentions)
Sc 22514, by Santa Cruz Biotechnology (1 mentions)
Ab9722, by Abcam (1 mentions)
Ab108362, by Abcam (1 mentions)
5 protocols using fluorchem is 8900
1
Western Blot Analysis of Hypoxia Markers
2
Protein Expression Analysis by Western Blot
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The total protein was collected after 48h by RIPA buffer containing protease inhibitor cocktail (Thermo fisher, USA). Whole cell protein extracts were boiled with SDS sample buffer and separated on 12% SDS-PAGE followed transferred onto a PVDF membrane (Roch, USA). The membranes were blocked in 5% nonfat milk for 1 h, incubated with antibodies against PARP (1:1000, CST, USA), p62 and LC3 (1:1000, CST, USA) and β-actin (1:1000, Santa Cruz Biotechnology, USA), respectively, according to the manufacturer's recommendations. The membranes were then washed and incubated with secondary antibody coupled to horseradish peroxidase for 2 h. The proteins were detected with western blot luminol reagent (Santa Cruz Biotechnology, USA) using a FluorChemTM IS-8900 (Alpha Innotech, USA).
3
Western Blot Analysis of PD-L1 and PD-1
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Protein samples (40 µg) were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with subsequent electrotransfer to nitrocellulose membrane (Bio-Rad-System, Germany). The membranes were blocked in TRIS-buffered saline with 0.1% tween containing 5% dry milk and the primary antibodies were added and incubated at 4 °C for 24 h. The antibodies were as follows: PD-L1, host rabbit (#13,684, Cell signaling); PD-1, host goat (AF1086, R&D Systems); cytoplasmic β-actin, host mouse (#MAK6019 Linaris GmbH). Then, the respective secondary anti-host antibodies coupled with horseradish peroxidase were added for band detection with enhanced chemiluminescent luciferase kit (Thermo Scientific, Rockford, USA) by an imager system (Fluorchem IS-8900, Alpha Innotech, San Leandro, USA).
4
Western Blot Analysis of Immune Signaling Proteins
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Protein samples (40 µg, determined by Pierce BCA Protein Assay (ThermoFisher, Scientific, Darmstadt, Germany)) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (gradient gel 4–20%) and transferred to nitrocellulose membranes by electro-blotting (Bio-Rad, Munich, Germany). The membranes were blocked at room temperature for 1.5 h in TRIS-buffered saline with 0.1% Tween containing 5% dry milk, and then the primary antibodies were added and incubated at 4 °C for 24–48 h. The antibodies were as follows: PD-L1 #13684, PD-L2 #82723, STAT1 #9172, JAK1 #3344, phospho-JAK1 #74129, IRF1 #8478, JAK2 #3230, phospho-JAK2 #3771; host rabbit (Cell Signalling Technology Europe, Frankfurt a.M., Germany); β-actin (#MAK6019); host mouse (Linaris, Dossenheim, Germany). Then, secondary antibodies against rabbit (Linaris) or mouse (Thermo Fisher) coupled with horseradish peroxidase were added for band detection with enhanced chemiluminescent luciferase kit (Thermo Scientific, Rockford, IL, USA) by an imager system (Fluorchem IS-8900, Alpha Innotech, San Leandro, CA, USA).
5
Western Blot Analysis of Inflammasome Proteins
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Hearts were homogenized in RIPA lysis buffer (9806; Cell Signaling). Protein concentration was determined by BCA kit assay (23225; Pierce Biotechnology). An equal amount of protein (30 μg) was separated by sodiumdodecyl sulphate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio‐Rad). Subsequently, the membrane was blocked with 5% nonfat dry milk or 3% bovine serum albumin (37520; Thermo Fisher Scientific) in TBST for 1 h (room temperature). Primary antibodies for NLRP3 (ab4207; Abcam, 1:500), ASC (sc22514; Santacruz, 1:500), caspase‐1 (ab108362; Abcam, 1:500), IL‐1β (ab9722; Abcam, 1:500), eNOS (#32027; Cell Signaling, 1:500), NOX2 (ab129068; Cell Signaling, 1:1000), NOX4 (ab133303; Abcam, 1:5000), and β‐actin (sc47778; Santa Cruz Biotechnology, 1:2000) were incubated at 4° overnight, and those were matched to their corresponding horseradish peroxidase‐conjugated secondary antibodies for 1 h (room temperature). Blots were developed with the enhanced chemiluminescence (RPN2232; GE Healthcare Life Science), scanned with the densitometer (FluorChem IS8900; Alpha Innotech, Santa Clara, CA), and quantified using NIH‐ImageJ software. Data were normalized with the corresponding internal reference β‐actin. Relative value was normalized to wild‐type control, LF‐SED.
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