Speedvac

Manufactured by Waters Corporation

Sourced in United States

The SpeedVac is a laboratory instrument designed for sample concentration and solvent removal. It utilizes vacuum evaporation to efficiently concentrate samples by removing solvents and other volatile components. The SpeedVac operates based on the principle of reduced pressure and controlled heating to facilitate the evaporation process.

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SpeedVac Vacuum Concentrator (3 citations)

10 protocols using speedvac

Breast Cancer Xenograft Proteomics Workflow

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Approximately 100 mg of each of the cryopulverized breast cancer xenograft tissues^{43 (link)} were sonicated in 1.5 mL of lysis buffer (8 M urea, 0.8 M NH₄HCO₃, pH 8.0). Protein concentrations of the tissue lysates were determined by a BCA assay (Pierce, Rockford, IL). Proteins were reduced with 10 mM TCEP for 1 h at 37 °C and subsequently alkylated with 12 mM iodoacetamide for 1 h at room temperature in dark. Samples were then diluted 1:4 with deionized water and digested with sequencing-grade modified trypsin (Promega, Madison, WI) at a 1:50 enzyme-to-substrate ratio. After 12 h of digestion at 37 °C, another aliquot of the same amount of trypsin was added to the samples and the samples were further incubated at 37 °C overnight. The digested samples were then acidified with 10% trifluoroacetic acid to pH <3. Tryptic peptides were desalted on reversed-phase C18 SPE columns (Waters, Milford, MA) and dried using a Speed-Vac.

Zhou J.Y., Chen L., Zhang B., Tian Y., Liu T., Thomas S.N., Chen L., Schnaubelt M., Boja E., Hiltke T., Kinsinger C.R., Rodriguez H., Davies S.R., Li S., Snider J.E., Erdmann-Gilmore P., Tabb D.L., Townsend R.R., Ellis M.J., Rodland K.D., Smith R.D., Carr S.A., Zhang Z., Chan D.W, & Zhang H. (2017). Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues. Journal of proteome research, 16(12), 4523-4530.

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ECM Extraction and Proteomic Analysis

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The ECM was mechanically detached from the 75 cm²-cell culture flask using a cell scraper in 2 ml of HBSS +/+, centrifuged at 16,000x g for 5 min, washed with 1 ml of HBSS +/+, centrifuged, and dried in a Speed-Vac (Savant) for 15 min. The ECM pellet was then processed as described previously [20 ,21 (link)]. Briefly, the ECM pellet was resuspended and reduced in a solution of 8 M urea, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol at pH 8 under agitation at 37°C for 2 h. After cooling, cysteines were alkylated by adding iodoacetamide at a final concentration of 25 mM for 30 min. The ECM sample was then diluted to 2 M urea, 100 mM ammonium bicarbonate (pH 8), and deglycosylated with PNGaseF (2000 U, New England BioLabs, Ipswich, MA) for 2 h under agitation at 37°C, followed by digestion with Lys-C (Wako Chemicals USA, Richmond, VA), at a ratio of 1:100 enzyme:substrate, under agitation at 37°C for 2 h. Final digestion was done using trypsin (Sequencing Grade, Promega, Madison, WI), at a ratio of 1:50 enzyme:substrate, under agitation at 37°C overnight, followed by a second aliquot of trypsin, at a ratio of 1:100 enzyme:substrate, and an additional 2 h of incubation. Digests were acidified and desalted using 30mg HLB Oasis Cartridges (Waters Corp., Milford, MA) eluted with 50% acetonitrile with 0.1% trifluoroacetic acid (TFA), followed by concentration in a Speed-Vac.

Ragelle H., Naba A., Larson B.L., Zhou F., Prijic M., Whittaker C.A., Del Rosario A., Langer R., Hynes R.O, & Anderson D.G. (2017). Comprehensive proteomic characterization of stem cell-derived extracellular matrices. Biomaterials, 128, 147-159.

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Exosomal Protein Isolation and Digestion

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Isolated urine exosomal proteins were resuspended in 100mM triethylammonium bicarbonate (TEABC; pH 8) containing 6 M Urea, 5 mM EDTA, and 2% SDS. The proteins were chemically denatured with 10 mM DTT for 20 min at 60°C, and alkylated with 50 mM iodoacetamide for 20 min at 25°C [15 (link)]. The denatured proteins were mixed with 30% acrylamide/bisacrylamide solution, 10% ammonium persulfate and tetramethylethylenediamine. The resulting gel was cut, then the gel was washed three times with 25 mM TEABC containing 50% acetonitrile (ACN). Trypsin digestion was performed in 25 mM TEABC overnight at 37°C. The peptides were extracted from the gel through exchange with two extraction buffers consisting of 0.1% formic acid (FA) in 25 mM TEABC or 0.1% FA in 50% ACN [16 (link)]. The buffer was dried in a SpeedVac and desalted by HLB cartridge (Waters, Milford, MA, USA).

Lim J.H., Lee C.H., Kim K.Y., Jung H.Y., Choi J.Y., Cho J.H., Park S.H., Kim Y.L., Baek M.C., Park J.B., Kim Y.H., Chung B.H., Lee S.H, & Kim C.D. (2018). Novel urinary exosomal biomarkers of acute T cell-mediated rejection in kidney transplant recipients: A cross-sectional study. PLoS ONE, 13(9), e0204204.

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Phosphatase and Kinase Assay Protocol

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Peptide digests were prepared as described above. Samples were then resuspended in 100 μL of Lambda phosphatase reaction buffer (50 mM Hepes, 100 mM NaCl, 2 mM DTT, 1 mM MnCl₂, pH 7.5). Lambda phosphatase (New England Biolabs P0753S) was added to each sample (2800–3000 units) and incubated overnight at 30 °C. The phosphatase was deactivated by heating at 75°C for 10 min. Samples were incubated in a kinase assay reaction buffer (100 mM Tris–HCl pH 7.8, 10 mM MgCl₂, 5 mM DTT, 2 mM ATP) containing the purified recombinant kinase (approximately 5000 units; estimated by in vitro kinase reaction with the recombinant, purified enzyme and relative quantification of bands via PepTag Nonradioactive protein kinase assays; Figure S2) at 30 °C for 90 min. Reactions were quenched by the addition of 0.5% TFA to a pH below 3. The samples were then desalted using Waters HLB cartridges and concentrated to dryness using a Speed-Vac.

Geller A.I, & Freese A. (1990). Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase.. Proceedings of the National Academy of Sciences of the United States of America, 87(3), 1149-1153.

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Small-EV Protein Quantification Protocol

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Protein concentrations of small-EVs in HT-29, HCT-116, and CRL-1541 cells were determined using the BCA assay after cell lysis. Digestion was performed as described previously [24 (link)]. Briefly, 50 μg protein was resuspended in 100 mM triethylammonium bicarbonate (TEABC; pH 8) containing 6 M urea, 5 mM EDTA, and 2% SDS. The proteins were chemically denatured by adding 10 mM dithiothreitol and incubating at 60 °C for 20 min. Next, proteins were alkylated by adding 50 mM iodoacetamide at room temperature and incubating for 20 min. The protein solution was mixed with acrylamide/bisacrylamide solution (30% v/v, 29:1) containing 10% (w/v) ammonium persulfate and tetramethylethylenediamine. The gel was cut into small pieces and washed three times with 50 mM TEABC containing 50% acetonitrile (ACN). Proteolytic digestion was performed using trypsin (protein:trypsin = 50:1 w/w) in 50 mM TEABC and incubating overnight at 37 °C. The digested peptides were extracted from the gel by exchanging with two buffers: 0.1% formic acid (FA) in 50 mM TEABC and 0.1% FA in 50% ACN. The eluents were concentrated using a Speedvac and desalted using an HLB cartridge (Waters, Milford, MA, USA).

Lee C.H., Im E.J., Moon P.G, & Baek M.C. (2018). Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. BMC Cancer, 18, 1058.

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Quantitative Proteomics using 10-plex TMT

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10-plex TMT reagents were used to label the desalted peptides from each sample according to the manufacturer’s instructions (Thermo Fisher Scientific, San Jose, CA, USA). Internal reference sample was prepared by mixing all samples at equal amounts, and then was used in Channel 126 throughout the sample analysis. The labeling efficiency of TMT reagents was checked with an EASY-nLC 1200 system coupled to an Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). After labeling efficiency confirmation (TMT modification ratio > 95% for both lysine residue and peptide N-termini), the peptides labeled by different TMT reagents were mixed with equal contribution, dried using Speed-Vac, and desalted by SepPak C18 cartridges (Waters, Milford, MA, USA). In total, the samples were labeled into four batches in the TMT 10-plex experiment.

Wang Z., Li Y., Zhao W., Jiang S., Huang Y., Hou J., Zhang X., Zhai Z., Yang C., Wang J., Zhu J., Pan J., Jiang W., Li Z., Ye M., Tan M., Jiang H, & Dang Y. (2023). Integrative multi-omics and drug–response characterization of patient-derived prostate cancer primary cells. Signal Transduction and Targeted Therapy, 8, 175.

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Analysis of Recombinant Factor IX by Mass Spectrometry

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Transgenic FIX (157.5 μg) was reduced with 2 mM dithiothreitol (Sigma) at 56°C for 1 h and alkylated for 1.5 h at room temperature (in the dark) with 5 mM iodoacetamide (Sigma). The sample was diluted with 50 mM ammonium bicarbonate and then 2 μg of trypsin was added and incubated for 18 h at 37°C. After tryptic digestion, the sample was deglycosylated overnight at 37°C either with or without PNGase F. The peptide fragments were analyzed by HPLC (Waters, Milford, MA) with a reversed phase column (Biobasic-18, 5 μm, 300 Å, 4.6 mm × 250 mm, Thermo Electron). The eluted fractions were collected manually according to the absorbance at 214 nm. Collected samples were dried by SpeedVac and then subjected to analysis by the CapLC System (Waters) using a capillary column (75 μm i.d., 10 cm in length, MST, Taiwan) with a linear gradient of 30% to 80% acetonitrile containing 0.1% formic acid over 40 min. The separated peptides were online analyzed under positive survey scan mode on an ESI-Q-TOF (Micromass, Manchester, UK) instrument. All MS/MS spectra were processed using Mascot Distiller (Matrix Science, London, UK), and the resulting PKL files were searched using the Mascot search engine v2.2 (Matrix Science).

Lee M.H., Lin Y.S., Tu C.F, & Yen C.H. (2014). Recombinant Human Factor IX Produced from Transgenic Porcine Milk. BioMed Research International, 2014, 315375.

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Mitochondrial Proteome Sample Preparation

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The mitochondrial pellets from the percoll density gradient ultracentrifuge were suspended in lysis buffer (7 M urea, 2 M thiourea, 5mM DTT and the protease inhibitor cocktail). After sonication for 3 min (5 s intervals for every 2 s), the ultrasound assisted lysate was centrifuged at 20, 000 g for 20 min. The supernatant was collected, and concentration of the mitochondrial protein lysate was determined by the Bradford assay. The protein solution was reduced with 10 mM DTT at 37°C for 45 min, alkylated with iodoacetamide (30 mM) at room temperature for 45 min. Then the proteins were digested at pH 8.0 with 1:50 (w/w) trypsin (Promega, V5113) and incubated overnight at 37°C with shaking. The digested peptides were desalted using a SepPark C18 cartridge (Waters) and dried with a SpeedVac.

Zeng H.L., Yang Q., Du H., Li H., Shen Y., Liu T., Chen X., Kamal G.M., Guan Q., Cheng L., Wang J, & Xu F. (2017). Proteomics and metabolomics analysis of hepatic mitochondrial metabolism in alcohol-preferring and non-preferring rats. Oncotarget, 8(60), 102020-102032.

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Proteome Isolation and Trypsin Digestion

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The lysis buffer containing 8M urea and protease and phosphatase inhibitors were added into biological samples at a 1:10 (w/v) ratio. Samples were placed on ice and sonicated 1 min with a 3-s interval at amplitude 25%. Then, the lysate was reduced with 10 mM DTT at 37 °C for 1 h and alkylated with 20 mM iodoacetamide at room temperature in the dark for an additional 15 min. BCA assay (Thermo Fisher) was performed to determine the proteins concentration and around 100 μg of protein was then digested using trypsin at an enzyme to protein ratio of 1:100 (w/w) at 37 °C overnight. Digested peptides were desalted using C18 cartridges (Waters) and dried in SpeedVac. The concentrations of peptide mixture were measured by peptide assay (Thermo Fisher). Samples were lyophilized and stored at −80 °C.

Donahue K., Xie H., Li M., Gao A., Ma M., Wang Y., Tipton R., Semanik N., Primeau T., Li S., Li L., Tang W, & Xu W. (2022). Diptoindonesin G is a middle domain HSP90 modulator for cancer treatment. The Journal of Biological Chemistry, 298(12), 102700.

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Quantitative Proteomic Profiling of CHO Cells

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Two biological replicates of CHO cells ectopically expressing human EPO protein were cultured in suspension with and without 1,3,4-O-Bu3ManNAc treatment. The cells were pelleted and washed twice with PBS while the protein in the medium was concentrated and buffer exchanged by 10 KD filter. Protein was harvested from each sample with 10 mL 8 M urea in 1 M NH4HCO3 followed by two rounds of sonication with 40-60 cycles per round. Protein was reduced by 10 mM TCEP (Thermo Fisher Scientific), alkylated with 16.5 mM iodoacetamide (Sigma-Adrich), and digested with sequencing-grade trypsin (Promega) at 1:50 enzyme:substrate ratio. Peptides were desalted using a C18 solid phase extraction (SPE) cartridge (Waters) and dried down by SpeedVac.

Mertz J.L., Sun S., Yin B., Betenbaugh M.J., Yarema K.J., & Zhang H. (2020). Comparison of three glycoproteomic methods for the analysis of CHO cells treated with 1,3,4-O-Bu₃ManNAc.

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