Urea was purchased from GE Healthcare (LC, UK). Proteinase inhibitor cocktail was purchased from Roche (BS, CH). TMT (Tandem Mass TagTM) kits were purchased from Thermo Fisher Scientific (NJ, USA). Sequencing grade Trypsin/Lys-C was purchased from Promega (WI, USA). Antibodies: Anti-ACTB (GTX124213, GeneTex, CA, USA), anti-FN1 (ab32419, Abcam, Cambridge, UK), and anti-NOS3 (sc-376751, Santa Cruz, CA, USA). The other reagents were obtained from Sigma (MO, USA).
Sequencing grade trypsin lysc
Manufactured by Promega
Sourced in United States
Sequencing grade Trypsin/LysC is a high-quality protease enzyme used for protein digestion. It is suitable for applications requiring precision and reliability, such as mass spectrometry-based proteomics analysis.
Automatically generated - may contain errors
Lab products found in correlation
Urea, by GE Healthcare (2 mentions)
Anti actb gtx124213, by GeneTex (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Seramag carboxylate modified beads, by GE Healthcare (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Tmt tandem mass tagtm kits, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Ab39184, by Abcam (1 mentions)
Ab182422, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using sequencing grade trypsin lysc
1
Multiplex Proteomic Analysis of Cellular Processes
2
Axon Proteomics Sample Preparation Protocol
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Axons were harvested by the addition of lysis buffer (1% SDC, 0.1% SDS, 100mM TrisHCl ph 8.5, 10mM DTT, 1x protease inhibitor EDTA free). Samples were supplemented with 25 units Benzonase nuclease (Merck), and lysed in a Bioruptor (Diagenode) for 5 minutes (cycle 30/30, 4°C). Alkylation was performed by addition of 30 mM Chloroacetamide followed by incubation in the dark for 30 min. Protein clean-up, digestion and peptide clean-up were performed using a modified version of the ultrasensitive sample preparation protocol SP3 (Hughes et al., 2014 (link)). In brief, 5 μL of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag Carboxylate-Modified beads, GE Life Sciences) were added to each sample. Acidified acetonitrile was added to achieve a final fraction of organic solvent of 50%. Beads were incubated for 10 min to allow complete binding of proteins to the beads. Protein clean-up was performed by subsequent wash with 70% Ethanol and once with Acetonitrile. For digestion, 0.1 μg sequencing grade Trypsin/LysC (Promega) was added and digestion was performed at 37°C for 16 h. Peptides were eluted with 9 μL 5% DMSO. 1 μL 10% formic acid was added and samples were stored at −20°C prior to MS analysis.
3
Proteomic Analysis of Macrophage Markers
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The following reagents and kits were used in this study: urea (GE Healthcare, LC, UK); protease inhibitor cocktail (Roche, BS, CH); Tandem Mass TagTM kits (Thermo Fisher Scientific, NJ, USA); and sequencing grade Trypsin/Lys-C (Promega, WI, USA). The following antibodies were used in this study: anti-VSIG4 (ab56037, Abcam, Cambridge, UK), anti-CD163 (ab182422, Abcam), anti-TIMP3 (ab39184, Abcam), and anti-ACTB (GTX124213, GeneTex, CA, USA).
4
Ultrasensitive Axonal Protein Preparation
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Axons were harvested by addition of lysis buffer (1% SDC, 0.1% SDS, 100 mM TrisHCl ph 8.5, 10 mM DTT, 1x protease inhibitor EDTA free). Samples were supplemented with 25 units Benzonase nuclease (Merck), and lysed in a Bioruptor (Diagenode) for 5 min (cycle 30/30, 4°C). Alkylation was performed by addition of 30 mM Chloroacetamide followed by incubation in the dark for 30 min. Protein clean-up, digestion, and peptide clean-up were performed using a modified version of the recently developed ultrasensitive sample preparation protocol SP3 (Hughes et al., 2014 (link)). In brief, 5 μL of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag Carboxylate-Modified beads, GE Life Sciences) were added to each sample. Acidified acetonitrile was added to achieve a final fraction of organic solvent of 50%. Beads were incubated for 10 min to allow complete binding of proteins to the beads. Protein clean-up was performed by subsequent wash with 70% Ethanol and once with Acetonitrile. For digestion, 0.1 μg sequencing grade Trypsin/LysC (Promega) was added and digestion was performed at 37°C for 16 hr. Peptides were eluted with 9 μL 5% DMSO. 1 μL 10% formic acid was added and samples were stored at −20°C prior MS analysis.
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