The protein level of CYPs in plasma exosomes was determined by loading 5 μg of protein in 10% SDS polyacrylamide gel followed by western blotting using standard protocol. In brief, the proteins from the gel were transferred to a polyvinylidene fluoride membrane and blocked using Li-Cor blocking buffer (LI-COR Biosciences, Lincoln, NE). The membrane was incubated overnight with primary antibodies (GAPDH Rabbit Mab, Cell Signaling Technology, Danvers, MA; CYP1A1, CYP2E1, and β-actin, Rabbit Mab, Abcam, Cambridge, MA; CYP1B1, CYP2C9, CYP3A4, CPR, and CD63, Mouse Mab. Santa Cruz Biotechnology. Inc. Dallas, TX) at 4°C using appropriate dilution (1:200 – 1:500). After subsequent washing, the blots were incubated with corresponding secondary antibodies (Goat anti-Mouse Mab and goat-anti-Rabbit Mab, LI-COR Biosciences) for 1 hour at room temperature. The blots were scanned with Li-Cor Scanner (LI-COR Biosciences). CD63 (Santa Cruz) was used as an internal loading control for exosomal proteins.
Goat anti rabbit mab
Manufactured by LI COR
Sourced in Niger
The Goat-anti-Rabbit Mab is a secondary antibody produced by LI-COR. It is designed to bind to primary rabbit antibodies, enabling their detection and visualization in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by LI COR (3 mentions)
Goat anti mouse mab, by LI COR (3 mentions)
Licor scanner, by LI COR (3 mentions)
Gapdh rabbit mab, by Cell Signaling Technology (2 mentions)
Sod1 mouse mab, by Santa Cruz Biotechnology (1 mentions)
Catalase mouse mab, by Santa Cruz Biotechnology (1 mentions)
Catalase, by Proteintech (1 mentions)
Cyp2e1, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
3 protocols using goat anti rabbit mab
1
Exosomal CYP Protein Detection
2
Western Blot Analysis of Protein Expression
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To determine the expression of proteins of interest, 30 μg of proteins in 5% SDS were separated on a polyacrylamide gel (4% stacking, 10% resolving gel) at 150 V for 70 minutes. The proteins from the gel were transferred to a polyvinylidene fluoride membrane at 0.35 Amp for 90 minutes. The transferred blots were blocked with 5–10 mL of Li-Cor blocking buffer (LI-COR Biosciences, Lincon, NE) for 1 hour and incubated overnight with primary antibodies (GAPDH Rabbit Mab, 1:2000 dilution, Cell Signaling Technology, Danvers, MA; CYP1A1 rabbit Mab, 1:200 dilution, Abcam, Cambridge, MA; CYP3A4 Mouse Mab. 1:200 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX; SOD1 Mouse Mab, 1:1500 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX; Catalase Mouse Mab, 1:1200 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX) at 4°C. After subsequent washing, the blots were incubated with corresponding secondary antibodies (1:10000 dilution, Goat anti-Mouse Mab, LI-COR Biosciences, Lincon, NE; 1:10000 dilution, Goat anti-Rabbit Mab, LI-COR Biosciences, Lincon, NE) for 1 hour at room temperature. The blots were scanned with Li-Cor Scanner (LI-COR Biosciences, Lincon, NE) and the densitometry data obtained from Image Studio Lite version 4.0 were used to calculate the fold expression of the proteins. GAPDH was used as an internal loading control to normalize the expression of sample proteins.
3
Oxidative Stress Protein Analysis in MDM
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Equal amount of proteins (20 µg) from 14-days treated experiments of MDM were loaded on 10% acrylamide gel. After SDS-PAGE, the proteins were transferred to a PVDF membrane. The membrane was blocked in Li-Cor blocking buffer (LI-COR Biosciences; Lincon, NE) for 1 h and incubated with primary antibodies against CYP2E1 (1:500, Millipore; Billerica, MA), catalase (1:1000, Proteintech; Rosemont, IL), PRDX6 (1:400, Proteintech), SOD1 (1:500, Santa Cruz; Dallas, Texas), SOD2 (1:500, Santa Cruz), and β-actin (1:4000, Cell Signaling; Danvers, MA) at 4 °C overnight. The membrane was then washed and incubated with secondary antibodies: goat anti-mouse Mab (1:10,000, LI-COR Biosciences) and goat anti-rabbit Mab (1:10,000, LI-COR Biosciences). The blots were scanned with a Li-Cor Scanner (LI-COR Biosciences).
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