Smart digest kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SMART digest kit is a sample preparation tool designed to facilitate the digestion of proteins in biological samples. It provides a streamlined workflow for fast and efficient protein digestion, enabling subsequent analysis by mass spectrometry or other analytical techniques.

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Lab products found in correlation

Pierce detergent removal spin column, by Thermo Fisher Scientific (1 mentions) Speedvac, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Merck Group (1 mentions) Fastprep 96, by MP Biomedicals (1 mentions) Thermomixer c, by Eppendorf (1 mentions)

Close spelling variants of this product

Thermo Scientific™ SMART Digest™ kits, Thermo Scientific™ SMART Digest™ pepsin kit SMART Digest Trypsin Kit

5 protocols using smart digest kit

Protein Extraction and Fractionation

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Twenty microliters of isolated vesicles were mixed with 80 μl of RIPA buffer (Sigma Aldrich) and the total protein concentration was determined using Bicinchoninic Acid Microtiter Plate Assays (Pierce).
The detergent was first removed from the samples (50 μl of protein) with Pierce Detergent Removal Spin Column (ThermoScientific, Waltham, MA). The proteins were then digested into peptides using the SMART digest kit (ThermoScientific) and fractionated with the Pierce High pH Reversed–Phase Peptide Fractionation columns (ThermoScientific). Eight tubes per sample were generated, using different concentrations of acetonitrile. Extracted peptides were then completely dried in a SpeedVac (ThermoScientific).

Val S., Jeong S., Poley M., Krueger A., Nino G., Brown K, & Preciado D. (2017). Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology. Pediatric research, 81(6), 911-918.

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Protein Fragmentation for Mass Spectrometry

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Each purified protein was then proteolytically fragmented using the Thermo Scientific™ SMART Digest™ kit as per the manufacturer’s instructions. The peptide fragments were lyophilized and made up in 20 μl 2% v/v acetonitrile containing 0.1% v/v formic acid for mass spectroscopy analysis.

Lukman V., Odeyemi S.W., Roth R.L., Mbabala L., Tshililo N., Vlok N.M., Dewar M.J, & Kenyon C.P. (2020). Novel kinase platform for the validation of the anti-tubercular activities of Pelargonium sidoides (Geraniaceae). BMC Biotechnology, 20, 50.

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EGCG Trophoblast Cell Lysis

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The Be-Wo trophoblast cells (1 × 10⁶), treated with or without EGCG (control), were suspended in PBS before being sonicated five times on ice. After sonication, the cell lysate was then spun at 14,000× g for 15 min at 4 °C to remove any cell debris. The resulting supernatants were then processed using a SMART Digest Kit (Thermo Fisher Scientific, Waltham, MA, USA) and cleaned of salt with ZipTip C18 microcolumns (Merck Millipore, Billerica, MA, USA). The resulting peptides were dried using a Speed-Vac and stored at −20 °C.

Chen Y.C., Liao C.C., Shui H.A., Huang P.H, & Shih L.J. (2023). A Proteomics-Based Identification of the Biological Networks Mediating the Impact of Epigallocatechin-3-Gallate on Trophoblast Cell Migration and Invasion, with Potential Implications for Maternal and Fetal Health. Proteomes, 11(4), 31.

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Glycoprotein Characterization from Swab Samples

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Proteins from the swab samples were extracted and homogenized in water using a bead-beater (Fastprep-96; MP-Biomedicals). 0.625 µl plasma or 50 µg protein from swabs or skin homogenates were prepared for MS analysis using the SmartDigest Kit (Thermo Fisher Scientific). Samples were denatured at 90°C followed by digestion for 3.5 h at 70°C. The peptides were reduced using 50 mM tris(2-carboxyethyl)phosphine, alkylated with 100 mM iodoacetamide, and finally purified using SOLAμ HRP plates (Thermo Fisher Scientific). The peptide samples were dried in a vacuum centrifuge and dissolved in 100 µl of 50 mM sodium acetate buffer (pH 5) containing 50 mU Thermatoga maritima α-fucosidase (Megazymes). After incubation at 70°C for 14 h, the samples were purified a second time on SOLAμ HRP plates and dried in a vacuum centrifuge.

Naegeli A., Bratanis E., Karlsson C., Shannon O., Kalluru R., Linder A., Malmström J, & Collin M. (2019). Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis. The Journal of Experimental Medicine, 216(7), 1615-1629.

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Plasma Protein Depletion and Digestion

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One hundred microliter of plasma were precipitated by adding PEG 6000 to final concentration of 12% in order to deplete abundant plasma proteins. Depleted plasma protein concentrations (n = 6 per group, Table 1) were determined using a BCA assay (Pierce) and digested using a Thermo Scientific™ SMART Digest™ Kit. Briefly, 50 µg of protein was added to 150 µL of the proprietary SMART digestion buffer and loaded into a SMART digestion tube containing immobilized trypsin beads. Samples were incubated at 70 °C and 1400 rpm for 2 h on an Eppendorf Thermomixer® C. Supernatants were collected by centrifugation at 2500×g for 5 min. The samples were desalted using SOLAµ™ solid phase extraction plates (Thermo Scientific, UK). Following washes of 0.1% trifluoroacetic acid (TFA) and elution in 65% ACN, samples were dried and resuspended for MS analysis as described above.

Thorne A.M., Huang H., O‘Brien D.P., Eijken M., Krogstrup N.V., Norregaard R., Møller B., Ploeg R.J., Jespersen B, & Kessler B.M. (2020). Subclinical effects of remote ischaemic conditioning in human kidney transplants revealed by quantitative proteomics. Clinical Proteomics, 17, 39.

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