Water, methanol (MeOH) and acetonitrile (ACN; all HPLC-grade) were obtained from Merck (Darmstadt, Germany). Sequence-grade trypsin and resuspension buffer was purchased from Promega (Mannheim, Germany). Any other chemicals and proteins were obtained from Sigma-Aldrich (Munich, Germany from).
Resuspension buffer
Manufactured by Promega
Sourced in Germany
Resuspension buffer is a solution used to reconstitute and resuspend dry or lyophilized biological samples, such as proteins, DNA, or RNA. It is designed to maintain the stability and activity of the sample during the resuspension process.
Automatically generated - may contain errors
Lab products found in correlation
Sequencing grade trypsin, by Promega (3 mentions)
Iodoacetamide, by Bio-Rad (2 mentions)
Protease max solution, by Promega (2 mentions)
Saccharomyces cerevisiae enolase digest, by Waters Corporation (2 mentions)
Pbs solution, by Thermo Fisher Scientific (2 mentions)
Dithiolthreitol dtt, by Bio-Rad (2 mentions)
Sequence grade trypsin, by Promega (1 mentions)
Acetonitrile acn, by Merck Group (1 mentions)
Methanol meoh, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Glu1 fibrinopeptide b glu1 fib, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
5 protocols using resuspension buffer
1
HPLC-grade Solvent Preparation
2
Mass Spectrometry Protein Identification
3
Trypsin-Mediated Protein Digestion
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Proteins were dissolved
in 50 mM Tris–HCl, 1 mM CaCl2, pH 7.6, and adjusted
to a concentration of 30–32 μM. Five hundred microliters
of protein solution were mixed with 10 μg of trypsin (sequencing
grade trypsin, Promega, catalog number V5111) dissolved at 40 μM
in resuspension buffer (Promega), and the proteolysis setup was incubated
at 37 °C. At different time points, samples were taken, mixed
with 1:20 (v/v) protease inhibitor cocktail set III (Calbiochem),
and frozen at −20 °C. For each protein sample, 5 μg
were subjected to SDS-PAGE analysis. After Coomassie staining gels
were analyzed using ImageLab software (Bio-Rad) using the volume tool.
Band intensities were normalized to protein bands of the molecular
weight standard. Values were analyzed statistically using the two-tailed t-test for independent samples (the threshold for the declaration
of statistical significance: P value < 0.05).
in 50 mM Tris–HCl, 1 mM CaCl2, pH 7.6, and adjusted
to a concentration of 30–32 μM. Five hundred microliters
of protein solution were mixed with 10 μg of trypsin (sequencing
grade trypsin, Promega, catalog number V5111) dissolved at 40 μM
in resuspension buffer (Promega), and the proteolysis setup was incubated
at 37 °C. At different time points, samples were taken, mixed
with 1:20 (v/v) protease inhibitor cocktail set III (Calbiochem),
and frozen at −20 °C. For each protein sample, 5 μg
were subjected to SDS-PAGE analysis. After Coomassie staining gels
were analyzed using ImageLab software (Bio-Rad) using the volume tool.
Band intensities were normalized to protein bands of the molecular
weight standard. Values were analyzed statistically using the two-tailed t-test for independent samples (the threshold for the declaration
of statistical significance: P value < 0.05).
4
Peptide Identification Mass Spectrometry
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Dithiolthreitol (DTT) and iodoacetamide were
purchased from Bio-Rad (Hercules, CA). MS-grade water, acetonitrile,
and formic acid (99%) were obtained from EMD Chemicals (Gibbstown,
NJ). Sequencing-grade trypsin, resuspension buffer, and protease MAX
solution were purchased from Promega (Madison, WI). [Glu1]-fibrinopeptide B ([Glu1]-Fib) and Saccharomyces
cerevisiae enolase digest were obtained from Waters (Milford,
MA). PBS solution was purchased from Fisher (Fair Lawn, NJ).
purchased from Bio-Rad (Hercules, CA). MS-grade water, acetonitrile,
and formic acid (99%) were obtained from EMD Chemicals (Gibbstown,
NJ). Sequencing-grade trypsin, resuspension buffer, and protease MAX
solution were purchased from Promega (Madison, WI). [Glu1]-fibrinopeptide B ([Glu1]-Fib) and Saccharomyces
cerevisiae enolase digest were obtained from Waters (Milford,
MA). PBS solution was purchased from Fisher (Fair Lawn, NJ).
5
CSF Proteomic Peptide Digestion
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In-solution digestion was performed as previously described [20 ]. Briefly, 50 µL CSF aliquots were spiked with QCAL internal calibrator (50 fmol) and pepmix (0, 10, 15, 30 fmol). Samples were reduced by adding 19.5 µL 24.2 mM Tris(2-carboxyethyl)phosphine solution and heated at 55 °C for 1 h. For alkylation, 2.4 µL freshly prepared 400 mM iodoacetamide (IAA) were added to each sample followed by vortexing and a 30 min incubation in the dark. Trypsin (20 µg per vial; Promega) was dissolved in 100 µL of resuspension buffer (Promega), and an aliquot containing 2.6 µg of the enzyme was added to each sample. Samples were digested overnight at 37 °C.
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