Speed vac

Manufactured by Agilent Technologies

Sourced in United States

The Speed-vac is a laboratory evaporator that is used to rapidly remove solvents from sample solutions through the process of vacuum concentration. It provides a controlled environment for the efficient evaporation of liquids.

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Lab products found in correlation

Omix c18 pipet tips, by Agilent Technologies (3 mentions) Bradford assay, by Bio-Rad (1 mentions) Tsq vantage triple quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions) Q exactive plus quadrupole orbitrap, by Thermo Fisher Scientific (1 mentions)

4 protocols using speed vac

Protein Quantification and Digestion for LC-MS/MS

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The protein lysates prepared from SW480 and SW620 cells were quantified using Bradford assay (Bio-Rad) and combined at 1:1 ratio (by mass), washed with 8 M urea for protein denaturation, and then treated with dithiothreitol and iodoacetamide for cysteine reduction and alkylation, respectively. The proteins were subsequently digested with modified MS-grade trypsin (Pierce) at an enzyme/substrate ratio of 1:100 in 50 mM NH₄HCO₃ (pH 8.5) at 37 °C overnight. The peptide mixture was subsequently dried in a Speed-vac, desalted with OMIX C18 pipet tips (Agilent Technologies), and subjected to LC–MS/MS analysis in the PRM mode.

Miao W, & Wang Y. (2019). Targeted Quantitative Kinome Analysis Identifies PRPS2 as a Promoter for Colorectal Cancer Metastasis. Journal of proteome research, 18(5), 2279-2286.

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Protein Denaturation, Reduction, and Digestion

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Cell lysates were washed with 8 M urea for protein denaturation, and dithiothreitol and iodoacetamide for cysteine reduction and alkylation, respectively. The proteins were subsequently digested with modified MS-grade trypsin (Pierce) at an enzyme/substrate ratio of 1:100 in 50 mM NH₄HCO₃ (pH 8.5) at 37 °C overnight. The resulting peptide mixture was dried in a Speed-vac, desalted with OMIX C18 pipet tips (Agilent Technologies), and analyzed by LC−MS and MS/MS on a Q Exactive Plus quadrupole-Orbitrap or a TSQ Vantage triple-quadrupole mass spectrometer (Thermo Fisher Scientific).

Miao W, & Wang Y. (2019). Quantitative Interrogation of the Human Kinome Perturbed by Two BRAF Inhibitors. Journal of proteome research, 18(6), 2624-2631.

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Metabolite Extraction and Derivatization Protocol

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The extraction procedure was followed the optimal protocol as previously reported [18 (link)]. Before extraction with 1 mL of a chloroform:methanol:water (1:2.5:1) mixture, 1 mg of caffeine was added as the internal standard to each 200 mg of powdered samples. The extraction was conducted by sonication for 30 min at room temperature and the crude extract was centrifuged for 5 min at 16,000 g. Then, 500 μL of the methanol/water phase was transferred to a 2 mL clear crimp vial (Agilent, Santa Clara, CA, USA) and dried using a SpeedVac at 5000 g and 25 °C for 5 h. For derivatization, 80 μL of methoxyamine hydrochloride in pyridine (15 mg/mL) was added to each vial and incubated in a 30 °C oven for 90 min. Thereafter, 100 μL of BSTFA with 1% TMCS was mixed with the solution and kept in an oven at 60 °C for 15 min.

Lim D.K., Mo C., Lee J.H., Long N.P., Dong Z., Li J., Lim J, & Kwon S.W. (2017). The integration of multi-platform MS-based metabolomics and multivariate analysis for the geographical origin discrimination of Oryza sativa L.. Journal of Food and Drug Analysis, 26(2), 769-777.

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SILAC-Based Quantitative Proteomics

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The protein lysates prepared from the aforementioned light and heavy SILAC-labeled PC3 cells were combined respectively with the heavy and light SILAC-labeled PC3MLN4 cells at a 1:1 ratio (in mass based on quantification from Bradford assay). Tryptic peptides for LC-PRM analysis were generated following the filter-aided sample preparation (FASP) protocol.^{21 (link)} Approximately 50 μg of cell lysates was washed with 8 M urea for protein denaturation using a Microcon centrifugal filter with a molecular weight cutoff of 30 kDa, and the urea buffer was then removed by centrifugation at 10000 rpm. The ensuing denatured proteins were treated with dithiothreitol and iodoacetamide for cysteine reduction and alkylation, respectively. The proteins were subsequently digested with modified MS-grade trypsin (Pierce) at an enzyme/substrate ratio of 1/100 in 50 mM NH₄HCO₃ (pH 8.5) at 37 °C overnight. The resultant peptide mixture was dried in a Speed-vac, desalted with OMIX C18 pipet tips (Agilent Technologies), and subjected to LC-MS/MS analysis in the PRM mode. Samples from three replicates (two forward and one reverse SILAC labeling experiment) of lysates from the PC3/PC3MLN4 pair of prostate cancer cells were prepared for LC-PRM analyses.

Miao W., Yuan J., Li L, & Wang Y. (2019). Parallel-Reaction-Monitoring-Based Proteome-Wide Profiling of Differential Kinase Protein Expression during Prostate Cancer Metastasis in Vitro. Analytical chemistry, 91(15), 9893-9900.

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