Samples (secretomes, EV fractions and HeLa whole cells) were lysed with RIPA buffer and protease inhibitor cocktail or with Mammalian Cell & Tissue Extraction Kit (BioVision), per manufacturers’ instructions. Lysates were resuspended in protein loading dye (Laemmli sample buffer; Bio-Rad) with freshly added β-mercaptoethanol (10%; v/v; Sigma). Sample mixtures were subsequently boiled for 10 min at 90°C and run on 4–15% TGX stain-free precast gels (Bio-Rad). Proteins were transferred to nitrocellulose Immun-Blot® Low Fluorescence PVDF membranes (Bio-Rad) then washed. After blocking, blots were probed with the primary antibodies: anti-TSG-101 (Abcam), anti-ALIX (Millipore; kindly provided by Dr. Shi Hua Li at Emory University), or anti-FN-1 and anti-Cdc42 (Santa Cruz). After incubation, membranes were washed and subsequently probed with IRDye® 680RD goat anti-rabbit or IRDye® 680RD goat anti-mouse (Li-COR), followed by washing. Initial HeLa cells were kindly provided by Dr. Michael Koval (Emory University). Protein concentration was determined using the BCA assay process with Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific), following manufacturer’s instructions. Blots were developed using SuperSignal West Dura Extended Duration Substrate (Thermo-Fisher) and protein bands were detected using the ChemiDoc MP Imaging System (Universal Hood III model; Bio-Rad).
Mammalian cell tissue extraction kit
Manufactured by Abcam
The Mammalian Cell & Tissue Extraction Kit is a set of reagents and protocols designed for the efficient extraction and purification of proteins, DNA, and RNA from mammalian cells and tissues. The kit provides a simple and reliable method for sample preparation, enabling downstream applications such as Western blotting, PCR, and other molecular biology techniques.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tgx stain free precast gel, by Bio-Rad (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
Irdye 680rd goat anti rabbit, by LI COR (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Anti tsg101, by Abcam (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
P stat1, by Cell Signaling Technology (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anykd gradient, by Bio-Rad (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
2 protocols using mammalian cell tissue extraction kit
1
Western Blot Analysis of EV and Secretome Proteins
2
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total protein lysates were extracted from NFASC silenced and control cells using Mammalian Cell & Tissue Extraction kit (BioVision, Milpitas, CA) and membrane fractions were isolated using Mem‐PER™ Plus Membrane Protein Extraction Kit (ThermoFisher Scientific). Concentrations were determined by Reducing Agent Compatible BCA Protein Assay kit (Biovision). Total protein lysates (80 μg) or membrane fractions (15 μg) were separated by 10% or AnyKD gradient (Biorad, Hercules, CA) SDS‐PAGE as appropriate and transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA). To prevent non‐specific background binding, the membranes were incubated with 5% non‐fat milk in Tris‐buffered saline with 0.05% Tween‐20 (TBST) for 1 h at room temperature. Membranes were incubated with primary antibodies against p‐AKT, AKT, p‐ERK, ERK, p‐STAT1, STAT1 p‐STAT3, STAT3, (Cell Signaling Technology, Inc., Beverly, MA), NFASC (Origene, Rockville, MD), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) over night at 4°C. After washing three times with TBST, the membranes were incubated with HRP‐conjugated secondary antibody (Cell Signaling Technology) for 1 h at room temperature and proteins visualized by SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific).
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