Proteins were extracted using RIPA lysis buffer (ThermoFisher). Protein concentration was determined by the BCA Protein Assay Kit (Thermo Fisher, Waltham, MA). Proteins (40 µg) were run on 4–20% gradient gels and transferred onto PVDF membranes using a Trans-Blot Turbo transfer pack and Trans-Blo Turbo transfer system (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (LI-COR) and incubated with primary antibodies overnight at 4 °C. The primary antibodies were SNAI1 (1:500, 3895 s, Cell signaling), KLF17 (1:500, sc-398132, Santa Cruz), ITGA6 (1:500, sc-374057, Santa Cruz), MYC (1:500, 2272 s, Cell signaling), Cleaved PARP (1:500, 5625 s, Cell signaling), β-Actin (A-9) (1:500, sc-1616, Santa Cruz). p-AKT (1:500, #9217c, Cell signaling), p-ERK (1:500,), p-JNK (1:500, sc-6254, Santa Cruz). total-JNK (1:500,), ERK inhibitor U0126 (10 µM, 19–147, Millipore), JNK inhibitor SP 600,125 (10 µM, S1460, Selleckchem).
Trans blo turbo transfer system
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo Transfer System is a laboratory equipment used for the efficient transfer of proteins from polyacrylamide gels to membranes for further analysis. It provides a rapid and consistent method for transferring proteins, enabling researchers to save time and optimize their workflow.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse hrp conjugated antibody, by Cell Signaling Technology (2 mentions)
Anti cd9, by Thermo Fisher Scientific (2 mentions)
Anti cd81, by Thermo Fisher Scientific (2 mentions)
Anti cd63, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Jnk inhibitor sp600125, by Selleck Chemicals (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Snai1, by Cell Signaling Technology (1 mentions)
Sc 6254, by Santa Cruz Biotechnology (1 mentions)
Itga6, by Santa Cruz Biotechnology (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Santa Cruz Biotechnology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Las 4000 plus, by Fujifilm (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Anti emmprin, by R&D Systems (1 mentions)
Fusion solo s, by Vilber (1 mentions)
3 protocols using trans blo turbo transfer system
1
Western Blot Protein Analysis Protocol
2
Exosome Protein Characterization by Western Blot
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Exosomes (1 μg protein) were subjected to 10% SDS–PAGE and the separated proteins were transferred onto polyvinylidene difluoride membrane using Trans-Blo Turbo Transfer System (BIO-RAD, Hercules, CA, USA) according to the manufacture’s instruction. Then, the membrane was placed in PBS-T solution containing each primary antibody: anti-CD9 (1 : 1000; Life technologies, Carlsbad, CA, USA), anti-CD63 (1 : 1000; Life technologies), and anti-CD81 (1 : 1000; Life technologies). The blots were incubated with secondary antibody (anti-mouse HRP- conjugated antibody, 1 : 300; Cell Signaling). The bands were detected using an enhanced chemiluminescence using ECL prime (GE health care, little Chalfont, UK). Western blots were analysed using the Luminescent image analyzer LAS 4000-plus (Fuji, Tokyo, Japan).
3
Extracellular Vesicle Protein Analysis
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EVs (100 ng protein) were subjected to 10% SDS-PAGE and the separated proteins and EV removal CM were transferred onto polyvinylidene difluoride membrane using Trans-Blo Turbo Transfer System (BIO-RAD) according to the manufacture's instruction. Then, the membrane was placed in PBS-T solution containing each primary antibody: anti-CD9 (1:500; Life technologies), anti-CD63 (1:500; Life technologies), anti-CD81 (1:500; Life technologies), anti EMMPRIN (1:500; R&D Systems). The blots were incubated with secondary antibody (anti-mouse HRP-conjugated antibody, 1:5000; Cell Signaling Technology). The chemiluminescence was detected by Fusion Solo S (Vilber Lourmat)
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