OPSS-PEG-NHS (molecular weight 5000) was purchased from Nanocs (New York, NY, USA). Trastuzumab (Herceptin) was a generous gift from Dr Virginia Borges (UC Denver, Denver, CO, USA). LLO from Escherichia coli transfected with the LLO-pEt29-DP-E3570 plasmid, kindly provided by Dr Dan Portnoy (UC Berkeley, Berkeley, CA, USA), was purified by the method described previously13 (link), 14 (link) and stored in storage buffer (50 mM phosphate buffer, pH 6.0, 1 M NaCl and 1 mM EDTA) without dithiothreitol to preserve its activity. Polylysine hydrobromide (molecular weight 37 000; degree of polymerization: 177) and 2-iminothiolane-HCl (Traut's reagent) were purchased from Sigma Life Science (St Louis, MO, USA). CL-4B Sepharose used for the purification of the one-component complexes was purchased from Amersham Biosciences (Uppsala, Sweden). All other reagents, unless otherwise specified, were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA).
Sepharose cl 4b
Manufactured by Cytiva
Sourced in Sweden
Sepharose CL-4B is a gel filtration media used for the separation and purification of biomolecules. It is a cross-linked agarose-based resin with a bead size range of 45-165 μm and a fractionation range of 60,000-20,000,000 Da for globular proteins. The product is designed for use in column chromatography applications.
Automatically generated - may contain errors
Lab products found in correlation
Polylysine hydrobromide, by Merck Group (2 mentions)
2 iminothiolane hcl, by Merck Group (2 mentions)
Traut s reagent, by Merck Group (1 mentions)
Pd 10 column, by Cytiva (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Doxorubicin hydrochloride, by Merck Group (1 mentions)
Dspe peg2000, by Avanti Polar Lipids (1 mentions)
Cetuximab erbitux, by Merck Group (1 mentions)
Sivelestat sodium salt hydrate, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Gm6001, by Merck Group (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Tskgel ods 120t, by Tosoh (1 mentions)
5 protocols using sepharose cl 4b
1
Trastuzumab-LLO Conjugation Protocol
2
Trastuzumab-Listeriolysin O Conjugate Synthesis
3
Antibody-Conjugated Quantum Dot Nanocarriers
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The chemicals, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[18 (link)] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000] (DSPE-PEG2000-mal), were purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). Anti-EGFR antibody (cetuximab, Erbitux®) was purchased from Merck (Darmstadt, Germany). QDs were purchased from ZEUS (Hwaseong, Republic of Korea). The PD-10 column and sepharose CL-4B were purchased from Amersham Bioscience (Uppsala, Sweden). Ultra-4 30 K MWCO was purchased from Amicon (Millipore, Darmstadt, Germany). Cell Counting kit-8 (CCK-8) was supplied by Dojindo Laboratories (Kumamoto, Japan). Doxorubicin hydrochloride was purchased from Merck (Darmstadt, Germany). Vyrant DiO Cell-Labeling Solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
4
Purification of Recombinant EMILIN1 Protein
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Secreted recombinant EMILIN1 protein was obtained by constitutive expression in 293E cells, as previously described [24 (link)]. Briefly, the cells were expanded to mass culture and then maintained for 2 days in serum-free medium to allow accumulation of EMILIN1 in the cell supernatant. Partial purification was achieved by dialysis of the conditioned medium at 4°C against 0.1 M NaCl, 20 mM Tris/HCI, pH 6.8. A further purification step was achieved by chromatography on a DEAE-cellulose column and size exclusion chromatography using Sepharose CL 4B (1.0×90.0 cm column, Amersham Pharmacia Biotech). Fibronectin was purchased from Sigma-Aldrich.
NE was purchased from Calbiochem (324681, Merk Millipore) and MMP-14 from Giotto Biotech S.r.l. Sivelestat sodium salt hydrate was purchased from Sigma (S7198) and suspended in a 20 mg/ml stock solution. GM6001 (Chemicon, Merk Millipore) was used as a broad-spectrum matrix metalloproteinase (MMP) inhibitor.
A complete list of all antibodies used in this study, with their specificity and sources/manufacturers, is given in Supplementary Table S1.
NE was purchased from Calbiochem (324681, Merk Millipore) and MMP-14 from Giotto Biotech S.r.l. Sivelestat sodium salt hydrate was purchased from Sigma (S7198) and suspended in a 20 mg/ml stock solution. GM6001 (Chemicon, Merk Millipore) was used as a broad-spectrum matrix metalloproteinase (MMP) inhibitor.
A complete list of all antibodies used in this study, with their specificity and sources/manufacturers, is given in Supplementary Table S1.
5
Liposomal Entrapment Efficiency Determination
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The encapsulation efficiency of liposomes was examined after separating free PMX from liposomes by Sepharose CL-4B (Amersham Bioscience, Uppsala, Sweden) column chromatography. 27) (link) Entrapped PMX was then determined by lysis of liposomes with methanol. The PMX content was analyzed by a high performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with a C 18 column (TSKgel ODS120T, TOSOH Bioscience) of a 4.6 mm×150 mm size. Phosphate buffer-acetronitrile (80 : 20) was used as a mobile phase at flow rate of 1 mL/min, an injection volume of 5 µL and at a wavelength of 254 nm. The PMX concentration was determined from the calibration curve of PMX at various concentrations. The experiment was independently performed for 3 repeating samples per experimental group (n=3). Experimental and theoretical percentages of PMX loading were calculated from Eqs. 1 and 2, respectively:
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