Radioiodination of anti-CD93 mAb and rabbit isotype IgG (dissolved in 0.01% sodium azide, 59% PBS, 40% glycerol and 0.05% BSA, pH 7.2; cat. nos. ab134079 and ab172730; Abcam) with Na125I was performed according to the Iodogen solid-phase labeling method, as previously described (24 (link)). In brief, 10 µg anti-CD93 mAb was added to 100 µl phosphate buffer (PB; 0.05 M; pH 7.4), and then 12 megabecquerel (MBq; 12 MBq=300 mCi) Na125I was added. The mixture was incubated for 20 min at 37°C, and the reaction was subsequently terminated by adding 150 µl 0.05 M PB and incubating the mixture for 10 min at 37°C. The labeling compound was purified on a Sephadex G-25 gel filtration column (GE Healthcare Life Sciences) as previously described (25 (link)) and the labeling efficiency was calculated. A mixture of 0.9% saline and methanol at a volume ratio of 1:2 was used as an unfolding agent. The stability of 125I-anti-CD93 mAb in vitro was determined in 0.05 M PBS (pH 7.4) or in human serum. The 125I-labeled IgG isotype was used as a non-specific control tracer and was prepared in a similar method as aforementioned.
Sephadex g 25 gel filtration column
Manufactured by GE Healthcare
Sourced in Sweden
Sephadex G-25 is a gel filtration column used for the separation and purification of molecules based on their size. It is composed of cross-linked dextran beads that create a porous matrix, allowing smaller molecules to enter the pores while larger molecules are excluded. This size-based separation technique is commonly used for desalting, buffer exchange, and the separation of proteins, peptides, and other small biomolecules.
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Lab products found in correlation
Ab134079, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Streptavidin, by Jackson ImmunoResearch (1 mentions)
Dsu 9 81 microscope, by Olympus (1 mentions)
Annexin 5 cy5 apoptosis detection kit, by Abcam (1 mentions)
Metaxpress image analysis software, by Molecular Devices (1 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Osmometer, by Knauer (1 mentions)
Mal peg 5k sva, by Laysan Bio (1 mentions)
C18 ods a, by YMC (1 mentions)
8 protocols using sephadex g 25 gel filtration column
1
Radioiodination of Anti-CD93 mAb
2
Determining Peptide Molecular Weights
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Sephadex G-25 gel filtration column (GE Healthcare Bio-Science AB, Uppsala, Sweden) was used for estimating the MW of peptides present in the selected HC. The fractions (3 mL) were pooled and tested for ABTS-RS-A. The MW of the selected fraction was calculated in comparison with those of protein markers, including blue dextran (2 000 000 Da), insulin chain B (3495.89 Da), vitamin B12 (1355.4 Da), glycine–tyrosine (238.25 Da) and tyrosine (181.2 Da).1
3
Streptavidin-Biotin Ro09-0198 Staining
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Streptavidin (Jackson) was conjugated with biotinylated Ro09–0198 (provided by Dr. M. Umeda, Kyoto Univ., Japan) and separated from free biotinylated Ro09–0198 using a Sephadex G-25 gel filtration column (GE Healthcare). For staining of cell surface PE, cells were incubated with 50 μg/ml SA-Bio-Ro for 30 min in α-MEM containing 0.2% (w/v) fatty acid-free bovine serum albumin (BSA) (Sigma). The cells were washed with 20 mM HEPES, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 2.2 mM CaCl2, 0.8 mM MgCl2 and 13.8 mM glucose, and then fixed with 4% (w/v) paraformaldehyde in PBS for 30 min, followed by a 5-min permeabilisation with 0.1% (v/v) Triton X-100 in PBS. Immunostaining was performed with a FITC-conjugated anti-Streptavidin antibody (Vector Laboratories). F-actin was stained using Alexa Fluor 594-conjugated phalloidin (Molecular Probes). PS on the cell surface was detected with an annexin V-Cy5 apoptosis detection kit (BioVision) in accordance with the manufacturer’s protocol. Fluorescence microscopy was performed using a DSU-IX81 microscope (Olympus). The images were photographed with a C10600 digital camera (Hamamatsu Photonics). In some experiments, immunofluorescence intensity of cells cultured in 96-well plates was quantified with an ImageXpress Micro XL HCS (Molecular Devices) and MetaXpress Image Analysis software (Molecular Devices).
4
Liposome Preparation with pH Gradient
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To obtain the LMVs, LUVs with a total lipid concentration of 3 mM were prepared with Citrate Phosphate buffer (pH 5.0). To change the external pH environment, LUVs were separated through a Sephadex G-25 gel filtration column (GE Healthcare, Little Chalfont, UK) using Hepes buffer (pH 7.4) as elution buffer. To prevent vesicle burst during gel filtration chromatography, the osmolarity of the buffers was measured using an osmometer (Knauer, Berlin), and when necessary sucrose was added to the elution buffer in order to have identical internal and external buffer osmolarity. Liposomes with pH 5.0 in the internal medium and pH 7.4 in the external medium were recovered mainly in fractions 3 and 4 (1 mL each), as confirmed by absorption, fluorescence and dynamic light scattering measurements (data not shown). The liposomes were then diluted to approximately 0.2 mM lipid concentration.
5
Peptide-Conjugated Iron Oxide Nanoparticles
6
Purification and Antioxidant Activity
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PHPH-III (250mg/mL) was loaded onto a Sephadex G-25 gel filtration column (2.6 × 80 cm, GE Healthcare, Uppsala, Sweden), equilibrated with distilled water, and eluted with distilled water at a flow rate of 0.5 mL/min, each fraction monitored at 220 nm using an ultraviolet detector, and the freeze-dried fractions was determined using the cellular antioxidant activity assay.
7
Purification and Proliferation Assay
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The fraction purified in the previous step (400 mg) was dissolved in 2 mL ultrapure water and purified using the Sephadex G-25 gel filtration column (8.0 × 48 cm, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), which had been equilibrated previously with ultrapure water. Ultrapure water was used as an eluent at a flow rate of 1.0 mL/min; 4 mL of the eluate was collected per tube and detected at 280 nm. Three sub-fractions were collected and lyophilized for determining their cell proliferation activity toward RAW 264.7 cells at a concentration of 100 μg/mL.
8
Isolation and Purification of Angiotensin-Converting Enzyme Inhibitors
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PFMHs were fractioned using ultrafiltration tubes (3 kDa MWCO), and two fractions (>3 kDa and <3 kDa) were obtained. Following the ACEI activity assay, the fraction with the higher activity was dissolved in distilled water at 100 mg mL−1 and loaded onto a Sephadex G-25 gel filtration column (3.5 × 30 cm, GE Ltd., USA), followed by elution using ultrapure water at a flow rate of 1.0 mL min−1 and evaluated at 220 nm. The eluted fractions were collected and lyophilized, and their ACEI activity was subsequently measured. Afterward, the fractions showing the highest activities were pooled and further purified on a YMC-Pack ODS-A C18 semi-preparative column (250 × 10.0 mm) using linear gradient eluting conditions at a flow rate of 2.0 mL min−1. The mobile phase consisted of eluent A (0.1% v/v trifluoroacetic acid (TFA) in ultrapure water) and eluent B (pure methanol). Following the injection of 100 μL of sample solution into the C18 column, the elution gradient was performed as follows: 99% A (0–5 min), 99–39% A (5–65 min), 39% A (65–70 min), 39–99% A (70–75 min) and 99% A (75–80 min). The eluted fractions were monitored at 220 nm and the fractions that showed the strongest ACEI activities were pooled and sequenced.
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