Ten milliliters of harvested medium was mixed with 2 ml of ConA Sepharose (GE Healthcare Bio-Sciences) and rotated overnight at 4°C. The mixture was then loaded into a column (0.8 cm × 4 cm) (Bio-Rad Laboratories, Hercules, CA, USA) and equilibrated with equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4). The column was washed with 50 ml of equilibration buffer to remove non-glycosylated and O-glycosylated proteins, and bound N-glycoproteins were eluted with 0.3 M methyl-α-D-glucopyranoside. Absorbance at 280 nm was measured throughout the chromatographic process. Eluted fractions were concentrated 100 times using Vivaspin concentrators (10,000 molecular weight cut-off; Sartorius) and further desalted with 2-D Clean-Up Kits (GE Healthcare Bio-Sciences) and then subjected to 2-DE analysis.
2 d clean up kit
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Germany
The 2-D Clean-Up Kit is a laboratory equipment product designed to prepare protein samples for two-dimensional (2D) gel electrophoresis. The kit provides reagents and materials to remove interfering substances, such as salts, lipids, and detergents, from protein samples prior to 2D gel analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 d quant kit, by GE Healthcare (48 mentions)
Immobiline drystrip, by GE Healthcare (7 mentions)
Microsonicator, by Qsonica (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Vivaspin concentrator, by Sartorius (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Protein extraction buffer 5, by GE Healthcare (3 mentions)
Ettan tm 2 d quant kit, by GE Healthcare (3 mentions)
Cydye dige fluor minimal labeling kit, by GE Healthcare (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (3 mentions)
Cyanine3 (cy3), by GE Healthcare (3 mentions)
Coomassie plus kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Protease inhibitor mix, by GE Healthcare (3 mentions)
Dithiothreitol (dtt), by GE Healthcare (3 mentions)
Chaps, by Thermo Fisher Scientific (3 mentions)
Thiourea, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Vivaspin 10 000 molecular weight cut off concentrators, by Sartorius (2 mentions)
207 protocols using 2 d clean up kit
1
Glycoprotein Enrichment and Purification
2
Proteomic Analysis of Secreted Proteins
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The growth media were removed when the cells were approximately 80% confluent in a 75 cm2 flask. The cells were then washed three times with phosphate buffered saline (PBS) (Invitrogen, California, US) at pH 7.4, and incubated for an additional of 24 h in serum-free DMEM. After 24 h, the media were harvested and centrifuged at 2,000 x g to remove all cell debris and kept at -80oC. Before subjecting the harvested media to two-dimensional electrophoresis (2D-E), the media were concentrated 100-fold using Vivaspin 10,000 molecular weight cut-off concentrators (Sartorius, Goettingen, Germany) and desalted with 2D Clean-Up Kits (GE Healthcare Bio-Sciences, Uppsala, Sweden).
3
Comparative Secretome Analysis of MCF-7 and HMEpC
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Similar amount of MCF-7 and HMEpC cells line were cultured in separate flasks with FBS-containing growth media and human mammary epithelial cell growth medium, respectively. The growth medias were removed when cells were approximately 80% confluent (108 cells) in a 75 cm2 flask. The cells were then washed three times with phosphate buffered saline (PBS) and incubated for an additional 24 h in serum-free DMEM. Ten milliliter of serum-free media was subsequently harvested, centrifuged at 2,000 × g to remove debris, and stored at –80°C. Prior to 2D-E, the samples were concentrated 100-fold using Vivaspin 10,000 molecular weight cut-off concentrators (Sartorius) and desalted with 2-D Clean-Up Kits (GE Healthcare Bio-Sciences, Uppsala, Sweden).
4
Quantitative Proteomic Analysis of Wood Samples
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Total proteins of fresh wood samples from five different time-points (0, 2, 6, 12, and 24 h) and from three biological replicates were extracted using the ReadyPrep™ Protein Extraction Kit (Total Protein) (Bio-RAD, USA) following the manufacturer's protocol. In total, 1 g of wood sample was powdered in liquid nitrogen using a mortar and pestle. The sample was transferred into a 5 mL microcentrifuge tube containing 2 mL of 2-D rehydration/sample buffer I, 20 μL of ReadyPrep TBP reducing agent and 0.2% (w/v) of the final concentrate of ampholyte. Subsequent steps were carried out according to the manufacturer's protocol. Total protein concentration was determined using the Quick Start™ Bradford protein assay kit (Bio-Rad, USA) on a Nanophotometer™ (IMPLEN, Germany) and bovine serum albumin (Bio-Rad, USA) was used as a standard. Upon quantification, 5 mg of total protein was transferred into a new 1.5 mL microcentrifuge tube, precipitated using 2-D Clean Up Kit (GE Healthcare Life Science, UK), following the manufacturer's protocol, and stored at −20 °C prior to in-solution tryptic digestion.
5
Proteomic Profiling of Cerebrospinal Fluid
6
Urine Protein Purification for Analysis
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Urine samples were centrifuged at 3000 × g for 15 min at 4 °C, and the supernatant was collected, supplemented with protease inhibitor cocktail (Complete Mini; Roche Diagnostics, Indianapolis, IN, USA), and stored at −80 °C. Albumin was removed from urine using the ProteoPrep Immunoaffinity kit (Sigma-Aldrich, St. Louis, MO, USA), and urine proteins were precipitated using the 2D clean-up kit (General Electric (GE) Healthcare Life Sciences, Piscataway, NJ, USA), which also removed interfering substances. The purified protein pellet was obtained by centrifugation, and then re-suspended in triethyl ammonium bicarbonate (TEABC) buffer. Protein quantification was performed by the Bradford assays.
7
Protein Extraction and Mass Spectrometry Protocol
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We trypsinized the cells, washed them 3-times with ice-cold PBS, and resuspended them in Protein Extraction Buffer-V (GE Healthcare, http://www.gelifesciences.com ) (urea, thiourea, CHAPS) containing a 2% Protease Inhibitor Mix (GE Healthcare, http://www.gelifesciences.com ). We purified all samples using a 2-D Clean-Up Kit (GE Healthcare, http://www.gelifesciences.com ) following the manufacturer´s instructions. We determined protein concentrations using a 2-D Quant Kit (GE Healthcare, http://www.gelifesciences.com ), which was compatible with components of the Protein Extraction Buffer-V.
We performed the isoelectric focusing, equilibration, the second dimension, and the staining of gels as previously39 (link). The MALDI Mass Spectrometry and Protein identification were performed following the protocol described previously68 (link).
We performed the isoelectric focusing, equilibration, the second dimension, and the staining of gels as previously39 (link). The MALDI Mass Spectrometry and Protein identification were performed following the protocol described previously68 (link).
8
Isolation and Purification of Dirofilaria repens
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Adult parasites were obtained from subcutaneous tissue of dogs during surgical procedures; microfilariae were collected from blood samples of infected dogs received from the Small Animal Hospital, Warsaw, University of Life Sciences. Microfilariae were isolated from blood using a filtration method with additional washing steps with PBS. Dirofilaria repens adult worms were washed with PBS to remove debris and homogenized manually in lysis buffer, containing 8 M Urea, 4% CHAPS, and 40 mM Tris, while microfilariae were suspended in lysis buffer and homogenized using TissueLyser LT (Qiagen, Hilden, NRW, Germany) and manual homogenizer. Lysates were purified with the 2-D Clean-Up Kit (GE Healthcare, Chicago, IL, USA), in accordance with the manufacturer’s protocols.
9
Serum Protein Depletion for HCC
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The blood samples of 5 of the 57 HCC patients were collected into clean glass tubes without an additive and were allowed to clot at room temperature for 60 min. Serum was separated by centrifugation at 1000 x g for 30 min to remove the insoluble solids. Aliquots of serum were then stored at − 80 °C until use. The removal of albumin and IgG was performed using the ProteoPrep Blue Albumin Depletion kit (Sigma, St. Louis, MO, USA), according to the manufacturer’s instructions. The 2-D cleanup kit (GE Healthcare, UK) was used to remove impurities from the protein extraction prior to the determination of the sample concentration using the 2-D Quant kit (GE Healthcare).
10
Protein Extraction and Digestion Protocol
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Eluted proteins were titrated to physiological pH and precipitated with the 2D Clean-Up Kit (GE Healthcare, Piscataway, NJ). The precipitated protein was resuspended in 8 m urea, 25 mm ammonium bicarbonate (pH 8), reduced with 5 mm TCEP (pH 8.0) for 30 min at room temperature, and alkylated with 10 mm IAA for 30 min at room temperature in the dark. Proteins were digested overnight with Lys-C 1:100 (w/w) at 37 ° C, and subsequently with trypsin 1:50 (w/w) overnight at 37 ° C. Samples were dried to completeness in a SpeedVac and stored at −80 ° C until analysis.
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