In a typical antibody-photosensitizer conjugate synthesis, 1 mg of CD44 mAb was first dispersed in 1 ml of 1X PBS and then added with 100 μl of K2HPO4 buffer (pH = 9.0) and 159.2 μg of IR700 (81.6 nmol, 1 mM in DMSO). The mixture was kept at 4 °C for overnight, and then placed in ultra-centrifugal filter units (Millipore Amicon Ultra-0.5, 10 kDa, Billerica, MA) to remove the unbound IR700 molecules. The IR700-conjugated anti-CD44 monoclonal antibody obtained was abbreviated as CD44-IR700. Similarly, we obtained mouse IgG conjugated with IR700 as a non-target control, which was abbreviated as IgG-IR700. The concentration of antibody and dye/protein ratio was determined spectroscopically by measuring the absorbance of the conjugate at 280 nm and 689 nm. The extinction coefficients were 210,000 M−1cm−1 for CD44 mAb at 280 nm, and 165,000 M−1cm−1 for IR700 at 689 nm. The correction factor of IR700 at 280 nm was set to be 0.095. The purity of CD44-IR700 was checked with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) performed on a 4–20% gradient gel (Mini-PROTEAN TGX precast gels, BIO-RAD, Hercules, CA). Fluorescence from the gel was obtained from a Typhoon gel scanner (GE Healthcare Bio-Sciences, Piscataway, NJ), and the protein bands on the gel were stained with GelCode® blue stain reagent (Thermo Fisher Scientific, Grand Islands, NY).
Amicon ultra 0.5 10 kda
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra-0.5 10 kDa is a centrifugal filter device designed for rapid concentration and purification of macromolecules. It features a regenerated cellulose membrane with a 10 kDa molecular weight cutoff, allowing efficient separation of proteins, peptides, and other molecules from smaller components in the sample.
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4 protocols using amicon ultra 0.5 10 kda
1
Antibody-Photosensitizer Conjugate Synthesis
2
Encapsulation Efficiency of α-Tocopherol Nanoemulsion
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Based on the optimized loading efficiency studied above, the encapsulation efficiency of α-TOC was determined by ultrafiltration method using centrifugal filter tubes (Amicon Ultra-0.5, 10 kDa, Millipore, Billerica, MA, USA), as described in previous studies [41 (link)]. The percentage of encapsulated α-TOC was calculated by the difference between the total amount of α-TOC added in the nanoemulsion and the amount of α-TOC encapsulated. The amount of α-TOC encapsulated was calculated as the initial α-TOC minus the free α-TOC that remained in the filtrate aqueous phase after ultrafiltration by centrifugation (15 min, 5000× g) of the nanoemulsion. Analysis of α-TOC was performed by the HPLC method reported below. The encapsulation efficiency was calculated using the following equation:
where EE is the encapsulation efficiency and Wa and Ws are the total weight of α-TOC added and weight of free α-TOC, respectively.
where EE is the encapsulation efficiency and Wa and Ws are the total weight of α-TOC added and weight of free α-TOC, respectively.
3
AID-mediated Gapped DNA Repair by Polη
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Close circular DNA gapped substrates with the lacZα-IGHV3-23*01 region as ssDNA were constructed as described previously (22 (link)). AID deamination reactions (30 μl total volume), containing GST-AID (100 ng), RNase (100 ng) and a gapped DNA substrate (500 ng) dissolved in a reaction buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol), were carried out at 37°C for 5 min and terminated by twice extracting the DNA product with phenol:chlorophorm:isoamyl alcohol (25:24:1). The deaminated gapped DNA (1 μg) were subjected to Polη gap-filling synthesis at 37°C for 2 h in a tube (100 μl total volume) containing 40 mM Tris–HCl (pH 8.0), 50 mM NaCl, 2.5% glycerol, 10 mM dithiothreitol, 2.5 mM MgCl2, 500 μM each of the four dNTPs and 300 ng of human Polη. Synthesis reaction was terminated and Polη was removed by twice extracting the DNA product with phenol:chlorophorm:isoamyl alcohol (25:24:1). AID and Polη treated DNA were desalted 4 times with H2O using Amicon Ultra-0.5 10 kDa (Millipore) centrifugal filter unit.
4
Construction of scFv Double-Gap Phagemids
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To make the scFv double-gap constructions, dsDNA phagemid pADL-20c and pADL20-scFv library were first digested with BglI and PvuI restriction enzymes, respectively. Linearized dsDNAs were cleaned and purified by QIAquick PCR purification kit (Qiagen). Linear pADL20c and pADL20-scFv (0.5 μg each in 45 μl of H2O) were heat denatured at 70°C for 5 min in separate PCR tubes and combined. After addition of 10 ul of 20× SSC buffer (3 M NaCl, 300 mM Sodium citrate, pH 7.0), the mixture was incubated at 60°C for 5 min and placed on ice. Double gapped DNA from eight tubes were pooled, desalted three times with H2O using Amicon Ultra-0.5 10 kDa (Millipore) centrifugal filter unit and stored in 1 mM Tris (pH 8.0) and 0.1 mM EDTA at −20°C. To verify the efficiency of gap formation, 10 pg of each linear pADL20c, pADL20-scFv, and the double gap constructs were used to transform 50 μl of TG1 electrocompetent cells by electroporation. The transformation mixtures were plated on LB plates in the presence of 100 μg/ml of ampicillin and TG1 bacterial colonies were counted after incubation at 37°C overnight.
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