Conditioned supernatants of multiple myeloma NCI-H929 cells were obtained by washing the cells in PBS and culturing them in Protein-Free Hybridoma medium for 72 h. Supernatants were collected and centrifuged at 12,000 × g for 30 min to remove cell debris. They were further concentrated using Amicon Ultra-0.5 centrifugal filter unit with ultracel-10 membrane (UFC501096, Milipore). For dot blot assay, 1 μl aliquots of 5 x concentrated samples were spotted on nitrocellulose membranes (Protean BA 85, GE Healthcare Life Sciences, Whatman). Released proteins were denatured, separated with 4–20% SDS-PAGE (Criterion TGX, Bio-Rad) and transferred to an Immuno-Blot TM polyvinylidene difluoride (PVDF) membrane (Bio-Rad) in western blot analysis. After blocking the membrane in 3% BSA dissolved in TBS, membranes were incubated 2 h at room temperature in 3% BSA with a primary polyclonal antibody GRP78 supplied with the GRP78-ELISA Kit (Biovendor). Afterwards, membranes were incubated with an anti-biotin HRP conjugated antibody (CST) diluted 1:1000. After washing, a chemiluminescent substrate (LumiGLO Reagent and Peroxide, Cell Signaling Technology) was added to the membrane, which was then exposed in the Chemidoc XRS station (Bio-Rad Laboratories).
Amicon ultra 0.5 centrifugal filter unit with ultracel 10 membrane
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane is a laboratory device designed for sample concentration and buffer exchange. It utilizes a polyethersulfone membrane with a molecular weight cut-off of 10 kDa. The device is suitable for volumes up to 0.5 mL and can be used with a standard laboratory centrifuge.
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Lab products found in correlation
Sodium hydroxide beads, by Merck Group (2 mentions)
Methyl iodide, by Merck Group (2 mentions)
2 5 dihydroxybenzoic acid 2 5 dhb, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Merck Group (2 mentions)
Pronase, by Roche (2 mentions)
Lumiglo reagent, by Cell Signaling Technology (1 mentions)
Peroxide, by Cell Signaling Technology (1 mentions)
Chemidoc xrs station, by Bio-Rad (1 mentions)
Criterion tgx, by Bio-Rad (1 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (1 mentions)
Nonidet p 40, by Roche (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
N n dimethylformamide dmf, by Mallinckrodt (1 mentions)
Lc ms grade water, by Merck Group (1 mentions)
Iodomethane, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Mono q 5 50 gl column, by GE Healthcare (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
6 protocols using amicon ultra 0.5 centrifugal filter unit with ultracel 10 membrane
1
Quantifying Extracellular GRP78 in Multiple Myeloma
2
Gelatin Zymography for Cell-Conditioned Media
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The gelatin zymography protocol has been described elsewhere [44 (link)]. Equal numbers of cells were plated in 10 cm plates. Once the cells had attached, the media was replaced with half the normal volume of serum-free media. The cells were incubated with the media for 16-18 hours and the media was collected. LNCaP- and VCaP-conditioned media required concentration using Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (#UFC5010BK, Millipore, Billerica, MA) and 10 μl of concentrated, conditioned media were loaded into the gelatin zymography. For PC3M-conditioned media, 25 μl of non-concentrated media were loaded directly into the gelatin zymography. For HT1080-conditioned media, 10 μl of non-concentrated media were loaded directly into the gelatin zymography.
3
Mass Spectrometry Sample Preparation
4
Glycoprotein Analysis by Mass Spectrometry
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Guanidine chloride, glacial acetic acid, formic acid (FA), and chloroform (Bio Clinical Lab, Phillipsburg, NJ, USA). Dimethyl sulfoxide (DMSO), sodium hydroxide beads, iodomethane, methyl-deuterium iodide, Trypsin (TPCK-treated from bovine pancreas) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Glycanase (PNGase F) was purchased from Prozyme (Hayward, CA, USA). Meanwhile, MALDI-TOF/MS calibrant mixture (calibrant Standard II) 2,5-Dihydroxybenzoic acid (2,5-DHB) was purchased from Bruker Daltonics (Bremen, Germany). LC-MS grade water, acetonitrile, and methanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sep-pak C-18 E 3.0 cc cartridges Strata X SPE were purchased from Phenomenex (Torrance, CA, USA), while Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane was from EMD Millipore (Billerica, MA, USA).
5
Purification of NttHNL from N. tambanus
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NttHNL was extracted from N. tambanus tambanus in phosphate buffered saline (PBS; 1.47 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and then purified. After adding ammonium sulfate to 30% saturation, the crude extract was loaded onto a TOYOPEARL Butyl-650M column (Tosoh, Tokyo, Japan). The adsorbed proteins were eluted by a step-wise gradient of ammonium sulfate in PBS (20%, 10%, and 0% saturation). The active fractions were loaded onto a PD-10 column (GE Healthcare, Little Chalfont, UK) equilibrated with 10 mM Tris-HCl (pH 9.0). The eluted proteins were loaded onto a MonoQ 5/50 GL column (GE Healthcare), and eluted with a linear gradient of sodium chloride (0–100 mM) in the same buffer. The active fractions were pooled and concentrated using a centrifugal filtration device (Amicon Ultra 0.5 Centrifugal Filter Unit with ultracel-10 membrane; Merck Millipore, Billerica, MA, USA). The concentrated enzyme solution was applied to a Superdex 200 10/300 column (GE Healthcare) equilibrated with PBS.
6
Glycan Extraction and Characterization
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