Whole‐cell extracts from BMDCs or DCEVs were obtained utilizing ice‐cold radioimmune precipitation assay buffer (Beyotime) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The extracts containing 30–50 μg proteins were separated on 10% SDS‐PAGE and then transferred to polyvinylidene fluoride membranes. The membrane was blocked with Tris‐buffered saline Tween 20 buffer containing 5% skim milk and incubated with the following primary antibodies: antigen‐MHC II (Abcam, Cambridge, UK), CD81 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), heat shock protein (HSP)70 (Abcam), HO‐1 (StressMarq Bioscience, Victoria, CA). The samples were incubated overnight followed by the addition of their corresponding horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). The signals were visualized via enhanced chemiluminescence using a Thermo ECL kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions.
Thermo ecl kit
Manufactured by Thermo Fisher Scientific
The Thermo ECL kit is a chemiluminescent detection system designed for the sensitive and reliable detection of proteins on Western blots. The kit provides reagents necessary for the visualization of target proteins labeled with horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated secondary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Radioimmune precipitation assay buffer, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Mouse anti cd63 antibody, by Santa Cruz Biotechnology (1 mentions)
2 protocols using thermo ecl kit
1
Western Blot Analysis of BMDC and EVCV Proteins
2
Protein Expression Analysis of Cellular Exosomes
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Total protein extracted from cells, exosomes or lung homogenates was prepared and solubilized in ice‐cold RIPA buffer (Beyotime) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Extracts containing 30–50 μg of protein were separated by 10% SDS‐PAGE and then transferred to PVDF membranes. The membranes were blocked with Tris‐buffered saline‐Tween‐20 buffer containing 5% skim milk and incubated with the following primary Abs: mouse anti‐CD63 antibody (Santa Cruz), mouse anti‐Tsg101 antibody (Santa Cruz), mouse anti‐Semaphorin‐4D/CD100 (E8S8A) mAb (CST), rabbit anti‐MMP14 polyclonal antibody (Absin), mouse anti‐Plexin B2 antibody (R&D) or rabbit anti‐β‐actin polyclonal antibody (CST). The samples were incubated overnight, followed by the addition of a corresponding anti‐rabbit, anti‐sheep (R&D) or anti‐mouse IgG secondary Ab (CST). Signals were detected via enhanced chemiluminescence using a Thermo ECL kit (Thermo Fisher Scientific).
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