EV pellets were solubilized in GuHCl lysis buffer [6 M GuHCl, 100 mM Tris pH 8.5, 10 mM tris(2-car- boxyethyl)phosphine, 40 mM 2-chloroacetamide] and heated for 5 min at 95 °C. Lysates were cooled on ice for 10 min, sonicated (Branson probe sonifier 10% duty cycle, 3 times, 20 s), and heated again (95 °C for 5 min). Lysates were centrifuged for 30 min at 10,000g at 4 °C, and cleared supernatant was removed to a clean tube. GuHCl concentration was diluted to less than 0.75 M using 100 mM Tris pH 8.5, and the samples were incubated overnight at 37 °C with trypsin (1:50 w/w). The reaction was stopped by adding trifluoroacetic acid to a final concentration of 0.1%, and the peptides were desalted using C18 Sep-Pak cartridges.
Probe sonifier
Manufactured by Emerson
The Probe Sonifier is a laboratory equipment designed for the disruption of cells, tissue, and other biological samples through the application of ultrasonic energy. It features a compact and durable construction, and is capable of delivering consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Ms grade trypsin, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Sep pak, by Waters Corporation (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Prestoblue reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using probe sonifier
1
Proteomic Analysis of Extracellular Vesicles
2
Quantitative Proteomic Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Sample pellets were solubilized in 3× volume of GuHCl lysis buffer (6 M Guanidine-HCl, 100 mM Tris-Cl pH 8.5, 10 mM TCEP, 40 mM CAA, 6 mM CaCl2) and heated for 5 min at 75 °C. Lysates were cooled on ice for 10 minutes, sonicated (Branson probe sonifier, 10% power, 3 × 20 s On, 10 s Off cycles), and heated again at 75 °C for 5 min, followed by centrifugation for 30 min at 3500 × g and 4 °C. Cleared lysates were transferred to fresh tubes, diluted 8× with 100 mM Tris-Cl pH 8.5 and protein concentration was determined using BCA assay (Thermo Scientific). Equal amounts of proteins were digested overnight with MS-grade Trypsin (Thermo Scientific) with 1:50 (protein: enzyme) ratio at 37 °C with agitation (900 rpm on an Eppendorf thermomixer). Digested peptides were acidified with 10% TFA (final 1% v/v), vacufuged to remove TFE and desalted using Sep-Pak (Waters Corp).
3
Microsphere Cytotoxicity Evaluation
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