EVs were purified from approximately 50 mL of cell culture medium collected from U87, A549, MIA PaCa-2 and HT-29 cell lines via differential ultracentrifugation, followed by lysis in a lysis buffer as previously described [18 (link)]. Protein lysates were separated by 10% sodium lauryl sulphate polyacrylamide (SDS-PAGE) gel, and then transferred onto apolyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich). The membranes were blocked andincubated at 4°C overnight with either an anti-EpcamAb (R&D systems, MAB960) or an anti-tsg101 Ab (Abcam, ab1337586), as tsg101 is reported to be widely expressed in EVs and could be used for normalization [19 ,20 (link)]. After being incubated with a secondary antibody (anti-mouse IgG (HAF007, R&D systems) for anti-Epcam, anti-rabbit IgG (ab205718, Abcam) for anti-tsg101) for 1 h at room temperature, the signals were visualized using the LI-COR Image Studio lite imaging system. The relative Epcam levels in EVs from different cell lines were normalized to the expression of tsg-101.
Anti mouse igg haf007
Manufactured by R&D Systems
Sourced in United States
Anti-mouse IgG (HAF007) is a secondary antibody product designed for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassays and applications. It is a purified goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ab205718, by Abcam (1 mentions)
Image studio lite imaging system, by LI COR (1 mentions)
Ly228820, by Selleck Chemicals (1 mentions)
Anti goat igg haf109, by R&D Systems (1 mentions)
Anti rabbit igg haf008, by R&D Systems (1 mentions)
Anisomycin, by Enzo Life Sciences (1 mentions)
Sb202190, by Selleck Chemicals (1 mentions)
Anti gapdh 5g4, by HyTest (1 mentions)
2 protocols using anti mouse igg haf007
1
EV Protein Expression Analysis Across Cell Lines
2
Reagents and Antibodies for P38 Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
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P38 inhibitors were purchased as follows: LY228820 and SB202190 from Selleck Chemicals (Houston, TX, USA); Doramapimod from Medichem Express (Princeton, NJ, USA) and dissolved in DMSO. Anisomycin was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and also solved in DMSO. Primary antibodies were purchased from the following providers: anti-DKK-1 (AF1096), anti-p38α (AF8691) and anti-p38γ (AF1347) from R&D Systems, Inc. (Minneapolis, MN, USA); anti-HSP27 (#2402), anti-p-HSP27 (#2405), anti-p38 (#2121) and anti-p-38 (#9211) from Cell Signaling Technology, Inc. (Beverly, MA, USA); anti-GAPDH (#5G4) from HyTest Ltd (Intelligate, Turku, Finland). Secondary HRP-conjugated antibodies: anti-goat IgG (HAF109), anti-mouse IgG (HAF007) and anti-rabbit IgG (HAF008) where purchased from R&D Systems, Inc. Control antibody for in vitro experiments: polyclonal goat IgG (AB-108-C) from R&D Systems, Inc. Recombinant bone morphogenetic protein 2 (BMP-2) was purchased from Peprotech (Rocky Hill, NJ, USA) and solved in water containing 0.1% bovine serum albumin (BSA) as the carrier protein.
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