4D5scFv-PE40 was labeled with fluorescein isothiocyanate (FITC, Thermo Scientific) or DyLight650 NHS Ester (Thermo Scientific), that are widely used amino-reactive dyes. These dyes contain isothiocyanate or N-hydroxysuccinimide (NHS) ester, respectively, reacting with primary amines in proteins. Prior to labeling reaction 4D5scFv-PE40 was desalted on Sephadex G-25 column (PD SpinTrap G-25, GE Healthcare) equilibrated with borate buffer (400 mM H3BO3, 70 mM Na2B4O7, pH 8.0). For FITC labeling, the protein was incubated with 5-fold molar excess of FITC dissolved in DMSO (Thermo Scientific) for 2 h at room temperature in the dark. For DyLight650 labeling, the protein was incubated with 7-fold molar excess of DyLight650 dissolved in DMSO (Thermo Scientific) for 1 h at room temperature in the dark. To remove unbound dye the reaction mixture was then desalted on Sephadex G-25 column (PD SpinTrap G-25, GE Healthcare) equilibrated with PBS.
Pd spintrap g 25
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden
The PD SpinTrap G-25 is a laboratory centrifugal desalting column designed to remove small molecules from protein samples. It is a single-use device that utilizes size-exclusion chromatography to separate proteins from salts, buffers, and other small molecules.
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Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Exoglow membrane ev labeling kit, by System Biosciences (4 mentions)
Chelex 100 resin, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Celllight reagent bacmam 2, by Thermo Fisher Scientific (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
3 5 dimethoxy 4 hydroxycinnamic acid, by Tokyo Chemical Industry (1 mentions)
Ultraflextreme, by Bruker (1 mentions)
Vivaspin, by GE Healthcare (1 mentions)
Epr2417y, by Abcam (1 mentions)
Clone lm609, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti hsc70, by Enzo Life Sciences (1 mentions)
Aldehyde sulfate latex beads, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Dotap, by Avanti Polar Lipids (1 mentions)
Nbd dmpe, by Avanti Polar Lipids (1 mentions)
Pettrace cyclotron, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Complete mouse serum, by Jackson ImmunoResearch (1 mentions)
34 protocols using pd spintrap g 25
1
Fluorescent Labeling of 4D5scFv-PE40
2
Lipid Vesicles Fused with PD-1 EVs
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A lipid thin film composed of DOPC and DOPS at a 1:1 molar ratio was hydrated with 1 mg/mL Texas Red-labeled dextran (TR-Dex, 3000 MW) (Thermo Fisher Scientific) solution dissolved in 10 mM Tris-HCl (pH 7.5) and the solution was incubated overnight at 27 °C. The suspension was extruded through a 100 nm pore polycarbonate membrane using a mini-extruder. To remove free TR-Dex, the suspension was washed using a PD SpinTrap G-25 (GE Healthcare). The TR-Dex-encapsulated liposomes (100 µM lipid) and PD-1 EVs (30 µg/mL protein) were mixed and incubated for 30 min at 27 °C under acidic conditions (pH 4.5). The fusion reaction was stopped by the addition of pH 7.5 buffer in the same volume as the reaction solution. The suspension was extruded again through a 100 nm pore membrane. Late endosomes and lysosomes of HeLa cells were GFP-labeled using CellLight™ reagent BacMam 2.0 (Thermo Fisher Scientific). The TR-Dex-encapsulated PD-1 hybrid EVs diluted in Opti-MEM reduced serum medium (Thermo Fisher Scientific) were incubated with 1 × 104 HeLa cells for 4 h in the presence or absence of 100 nM bafilomycin A1. After washing with PBS three times, the cells were observed by a confocal laser scanning microscope, LSM780 (Carl Zeiss). The co-localization coefficient was calculated from the ratio of all Texas Red pixels to Texas Red pixels co-localized with GFP.
3
Quantifying NOTA Conjugation to Transferrin
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Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed using an ultrafleXtreme (Bruker Daltonics, Bremen, Germany) to examine the number of chelators conjugated with aTf. Non-conjugated- and chelator-conjugated aTf were desalted using PD Spin Trap G-25 (GE Healthcare Life Science). 3,5-Dimethoxy-4-hydroxycinnamic acid (Tokyo Chemical Industry Co.) at 10 mg/mL in 1:1 acetonitrile/H2O with 0.1% trifluoroacetic acid was used as the MALDI matrix. For each sample, measurements were repeated four times. The measured mass difference between aTf and NOTA-aTf was divided by the mass value of single NOTA, and the resulting values represented the average number of NOTA that were conjugated to aTf.
4
Ultrafiltration and Column Purification
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Ultrafiltration was performed as previously described (Iki et al. 2010 (link)) using Vivaspin (molecular weight cutoff 10,000; GE Healthcare). Column purification was performed using PD SpinTrap G-25 (GE Healthcare). In vitro translation reaction mixtures (140 µL) were passed through columns preequilibrated with TR buffer (30 mM HEPES [pH 7.4], 80 mM KOAc, 1.8 mM MgCl2, 2 mM DTT, 1 tablet/50 mL complete protease inhibitor [Roche]).
5
Pronase Digestion of Mouse Liver Tissue
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Pronase digestion was performed on mouse liver tissue samples, similarly to previously described procedure, with adequate modifications (29 (link)). Briefly, deep-frozen tissue samples were homogenized in 0.5 ml of ice-cold radioimmunoprecipitation assay (RIPA) buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, 0.1% sodium deoxycholate, and 0.1% SDS] containing 5 mM HPE-IAM. Lysates were centrifuged at 15,000g for 10 min at room temperature. Supernatant was recovered, and 150 μl was applied to PD SpinTrap G-25 desalting column (GE Healthcare) to remove the LMW fraction. HPE-IAM (5 mM) was immediately reintroduced to the filtrates. After BCA protein assay, protein levels were brought equal to 1 mg/ml and digested with pronase (3 mg/ml) in 50 mM sodium acetate buffer (pH 5.0) in the presence of 40 nM stable isotope–labeled internal standards for 7 hours at 37°C. After centrifugation, the supernatants were subjected to LC-ESI-MS/MS analysis as mentioned above.
6
Investigating Anti-HPA-1a mAb Effects on Trophoblasts
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A recently developed human recombinant anti-HPA-1a IgG1 mAb (clone 26.4) [16 (link)] was used to explore the effect on invasive trophoblast cells. Murine anti-human αVβ3 mAb, clone LM609 (Millipore, Billerica, MA) was used as positive control for cell functional studies. Sodium azide from LM609 sample was removed by buffer exchange with PBS using PD SpinTrap G-25 (GE Healthcare, Little Chalfont, UK). Integrin β3 was detected using murine mAb, clone SZ21, HPA-1-reactive [19 (link)] (Dako, Glostrup, Denmark) and rabbit mAb, clone EPR2417Y (Abcam, Cambridge, UK). Alexa Fluor 488-conjugated goat anti-mouse and goat anti-human antibodies (Invitrogen, Carlsbad, CA) were used as secondary antibodies in flow cytometry experiments. Human myeloma plasma IgG1 (Sigma) and murine IgG1 (Beckman Coulter, Brea, CA) were used as isotype controls. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Thermo Scientific, Waltham, MA) was used as a detection antibody in the western blot experiment.
7
Antibacterial Assay of Epidermal Extracts
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Epidermis isolated from 19.5 dpc mice (n = 3) was placed on ice and minced in 0.5 ml 1 M HCl. The specimens were homogenized using a mixer mill MM 300 (Retsch Technology GmbH, Haan, Germany) and were incubated at 4°C for 24 h under rotation. After centrifugation (10,000 x g) at 4°C for 10 min, the supernatant was lyophilized and dissolved in 0.3 ml 5 mM MOPS buffer (pH 7.0). The solution was desalted using a PD SpinTrap G-25 (exclusion limit: Mr. 5,000) (GE Healthcare Bio-Sciences, Pittsburgh, PA) and 0.2 μg/μl protein solution was subjected to colony forming unit (CFU) assays as described by Sørensen et al. [11 (link)]. Bacterial suspensions of 0.2 ml 1.0 x 106 cfu/ml Staphylococcus aureus (DSM 346 strain) or E. coli (K-12 strain) were mixed with 0.1 ml diluted protein solutions or 5 mM MOPS buffer (pH 7.0) as a control and were incubated at 37°C for 3 h. Serially diluted bacterial cultures were plated on Tryptic Soy agar plates, and after incubation at 37°C for 24 h, the number of colonies was counted. For statistical analysis of multiple comparison, one-way ANOVA and Bonferroni post hoc test was used, and a P<0.05 is considered a significant difference.
8
Refolding of His-tag-attached HT cyt c552
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Refolding of His-tag-attached HT cyt c552 was performed using a desalting gel column (PD SpinTrap G-25, GE Healthcare) at 4 °C as reported previously19 (link). Additional details on the experimental procedures are provided in supplementary information .
9
Exosome Characterization Protocols
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DOPC, DOPS, and PEG-DSPE, were purchased from NOF Corp. Ltd. (Tokyo, Japan). DOTAP (chloride salt), NBD-DMPE, and rho-DMPE were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Anti-Hsc70 (Anti-Hsc70 rat monoclonal antibody [1B5]), anti-HER2 (anti-ErB2), anti-CD9, anti-phosphorylated HER2 (p-Neu[Tyr1248]), phycoerythrin (PE)-conjugated mouse anti-human CD63, horseradish peroxidase (HRP)-conjugated goat-anti rabbit-IgG, and PE-conjugated mouse IgG1 were purchased from Enzo Life Sciences (Farmingdale, NY, USA), abcam (Cambridge, UK), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Biolegend (San Diego, CA, USA), and BD Bioscience (San Jose, CA, USA), respectively. ECL Western Blotting Detection Reagent and PD SpinTrap G-25 were purchased from GE Healthcare Japan (Tokyo, Japan). Aldehyde/sulfate latex beads (4% w/v, 4 μm) and stain buffer (FBS) were purchased from Life Technologies (Carlsbad, CA, USA) and BD Bioscience (CA, USA), respectively. CFSE was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan).
10
Radiolabeling of Antibodies with 64Cu
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