Amicon ultra 0.5 ml 10k

Manufactured by Merck Group

The Amicon Ultra 0.5 mL 10k is a centrifugal filter device used for the concentration and desalting of samples. It has a 10 kDa molecular weight cut-off membrane and a maximum sample volume of 0.5 mL.

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Amicon Ultra 0.5 mL 10K cutoff desalting columns Amicon Ultra-0.5 mL 10K centrifugal filters Amicon Ultra 0.5 mL Amicon Ultra 10K Amicon Ultra-0.5 Amicon Ultra

7 protocols using amicon ultra 0.5 ml 10k

Synthesis of PEG-TRAIL-Cy5 Conjugates

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For the synthesis of PEG-TRAIL-Cy5 conjugates, the TRAIL mutant N109C (1 mg/mL) in PBS (pH 7.4) was treated with TCEP HCl (10 eq) at 37 °C for 1 h to disrupt intermolecular disulfide bond. To the reduced N109C at room temperature was added the methoxyl-PEG-maleimide (mPEG-MAL, 5000 Da) (2 eq/SH group) in PBS. After 40 min, sulfo-cyanine5-maleimide (water-soluble Cy5, a near- IR fluorophore) (4 eq/SH group) dissolved in PBS was added to the above reactions for the co-modification of TRAIL mutant with PEG and Cy5. For the preparation of BSA-Cy5, BSA (1 mg/mL) was treated with TCEP HCl (10 eq) at 37 °C for 1 h to disrupt intermolecular disulfide bond. To the reduced BSA at room temperature was added sulfo-cyanine5-maleimide (2 eq/SH group), and reacted for 40 min. All the reactions were kept from light.
For the subsequent in vivo distribution expriments, BSA-Cy5 and PEG-N109C-Cy5 conjugates were desalted by centrifugal ultrafiltration (Amicon Ultra-0.5 mL 10K, Millipore), and then sterile filtered. Protein and drug concentrations were determined by spectral analysis. Due to the similar molecular weight of Cy5 (803 g/mol) with vcMMAE (1316.63 g/mol), Cy5-maleimide could be an ideal fluorescent marker without deviating from structural effect of conjugating PEG-N109C with MMAE.

Pan L.Q., Zhao W.B., Lai J., Ding D., Wei X.Y., Li Y.Y., Liu W.H., Yang X.Y., Xu Y.C, & Chen S.Q. (2015). Hetero-modification of TRAIL trimer for improved drug delivery and in vivo antitumor activities. Scientific Reports, 5, 14872.

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Radiolabeling of Gold Nanoparticles

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In a 2 mL eppendorf, 100 µL of 0.4 M ammonium acetate (pH ≈ 7) were mixed with 5 µL of the desired AuNPs (5 mg/mL in DI water) (AuNP-TDOTA and AuNP-SPTyr8). Following the addition of 3 µL of ¹⁷⁷LuCl₃ in 0.04 M HCl (37–74 MBq), the mixture was heated at 70 °C. After cooling to RT, radio-TLC analysis showed that a radiochemical yield >95 % was obtained. The solution was filtered in a Millipore Amicon Ultra 0.5 mL 10k and the filtered gold nanoconstructs washed with H₂O (2 × 400 µL) and finally recovered from the filter in 150 μL of H₂O. Thereafter, radio-TLC analysis showed that ¹⁷⁷Lu-AuNP-TDOTA and ¹⁷⁷Lu-AuNP-SPTyr8 were obtained with a radiochemical purity >95 %.

Silva F., D’Onofrio A., Mendes C., Pinto C., Marques A., Campello M.P., Oliveira M.C., Raposinho P., Belchior A., Di Maria S., Marques F., Cruz C., Carvalho J, & Paulo A. (2022). Radiolabeled Gold Nanoseeds Decorated with Substance P Peptides: Synthesis, Characterization and In Vitro Evaluation in Glioblastoma Cellular Models. International Journal of Molecular Sciences, 23(2), 617.

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Radiolabeling of Gold Nanoparticles

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20 µL of NP solutions (5 mg/mL in DI water) was mixed with 350 µL of 0.4 M ammonium acetate (pH ≈ 7). To this mixture, 250 µL of ⁶⁷GaCl₃ (0.1 M HCl) was added, followed by heating at 70 °C for 30 min. After cooling to room temperature, the reaction mixture was then filtered through a Millipore Amicon Ultra 0.5 mL 10k. The collected gold nanoconstructs were diluted in 150 µL of H₂O and their radiochemical purity assessed by ITLC-SG, using 6M HCl/MeOH (5:95) as an eluent. All nanoparticles were obtained with a final radiochemical purity >95%.

Silva F., Paulo A., Pallier A., Même S., Tóth É., Gano L., Marques F., Geraldes C.F., Castro M.M., Cardoso A.M., Jurado A.S., López-Larrubia P., Lacerda S, & Cabral Campello M.P. (2020). Dual Imaging Gold Nanoplatforms for Targeted Radiotheranostics. Materials, 13(3), 513.

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Radiolabeling of AuNPs with 67Ga

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In a 2 mL eppendorf, 350 µL of 0.4 M ammonium acetate (pH ≈ 7) were mixed with 20 µL of the desired AuNPs (5 mg/mL in DI water) (AuNP-TDOTA, AuNP-SP and AuNP-SPTyr8). Then, 250 µL of ⁶⁷GaCl₃ in 0.1 M HCl (37–74 MBq) were added. The mixture was heated at 70 °C for 30 min and, after cooling to RT, radio-TLC analysis showed that a radiochemical yield >95 % was obtained. The solution was filtered in a Millipore Amicon Ultra 0.5 mL 10k and the filtered gold nanoconstructs washed with H₂O (2 × 400 µL) and finally recovered from the filter in 150 μL of H₂O. Thereafter, radio-TLC analysis showed that ⁶⁷Ga-AuNP-TDOTA, ⁶⁷Ga-AuNP-SP and ⁶⁷Ga-AuNP-SPTyr8 were obtained with a radiochemical purity >95 %.

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Radiolabeling of Bombesin-Conjugated Gold Nanoparticles

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In a 2 mL eppendorf, 350 µL of 0.4 M ammonium acetate (pH ≈ 7) were mixed with 30 µL of AuNP-BBN-Pt1 (5 mg/mL in DI water) or 40 µL of AuNP-BBN-Pt2/AuNP-BBN-Pt3 (5 mg/mL in DI water). To this mixture, 250 µL of ⁶⁷GaCl₃ in 0.1 M HCl (20-70 MBq) were added. The mixture was heated at 70 °C for 30 minutes The solution was then filtered in a Millipore Amicon Ultra 0.5 mL 10k to recover the ⁶⁷Ga-labeled AuNPs (radiochemical yield > 95 %). The radiochemical purity of the collected gold nanoconstructs was assessed by radio-TLC (> 95 %), using an ITLC-SG support and CH₃OH/HCl 6M (95:5) as eluent system.

Silva F., Mendes C., D'Onofrio A., Campello M.P., Marques F., Pinheiro T., Gonçalves K., Figueiredo S., Gano L., Ravera M., Gabano E, & Paulo A. (2023). Image-Guided Nanodelivery of Pt(IV) Prodrugs to GRP-Receptor Positive Tumors. Nanotheranostics, 7(1), 22-40.

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Quantifying Hold-up Volumes in BWEC Devices

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Example 5

Hold-up volumes were evaluated with bind-wash-elute-concentrate (BWEC) devices and diaphragm caps. The devices were pre-washed with 1.5 ml BSA (1 mg/ml PBS) at 4000×g for 2 minutes, and then 0.5 ml BSA (1 mg/ml PBS) was added to each of the devices after assembling with a 0.5 μm filter device (AMICON ULTRA 0.5 ml 10K, available from EMD Millipore Corporation), followed by centrifugation for 15 minutes at 4000×g. The hold-up volumes were calculated by weight difference of the devices before and after actuating the diaphragm. The results are shown in FIG. 26, and demonstrate that actuation of the diaphragm results in the recovery of more than 1.5 μl of sample compared to no diaphragm.

US09897520B2. All-in-one sample preparation device and method (2018-02-20). EMD Millipore Corporation [US]. Inventors: Christopher A. Scott [US], Timothy K. Nadler [US], Kurt Greenizen [US], Louis Bonhomme [US], Sara D. Gutierrez [US], Masaharu Mabuchi [US], David Briggs [US], Phillip Clark [US], Ralph T. Scaduto [US].

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Bind-Wash-Elute Buffer Exchange Evaluation

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Example 6

Bind-wash-elute (BWE) devices were evaluated on buffer exchange. 50 μl of 10 mM Tris, pH 7.5, 1 M NaCl was distributed to a filter device (AMICON ULTRA 0.5 ml 10K, available from EMD Millipore Corporation) and assembled into exchange tubes and centrifuged at 4000×g for 15 minutes after adding 1.5 ml of 10 mM Tris, pH 7.5 to the exchange tube. The retentates were collected by reverse spin for 2 minutes at 1000×g and the final volume was adjusted to 100 μl with 10 mM Tris. Conductivities were measured after adding 4.9 ml Milli-Q water. For the 3-spin control, buffer exchange was carried out by three consecutive washes with 0.5 ml. FIG. 27 shows that bind-wash-elute performed equivalent to the 3-spin method despite only a single spin.

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