Supernatants with unbound proteins were removed and the beads re-suspended in bead wash buffer (1 mL). The beads were washed twice with bead wash buffer (1 mL) then transferred to clean 1.5 mL microcentrifuge tubes (ThermoFisher Scientific, Waltham, MA). After washing the beads with PBS (1 mL), hFXN-M and mFXN-M were eluted with 200 μL of elution buffer (100 mM acetic acid in 10% ACN) by shaking the beads in a thermal mixer (ThermoFisher Scientific, Waltham, MA) at 1000 rpm and RT for 1 h. Supernatants were then transferred to Sarstedt 2.0 mL LB microtubes and dried under a nitrogen flow using an N-Evap concentrator (Organomation, Berlin, MA).
1.5 ml microcentrifuge tube
Manufactured by Thermo Fisher Scientific
Sourced in United States
1.5 mL microcentrifuge tubes are small, cylindrical containers used for holding and processing small volumes of liquid samples in a laboratory setting. These tubes are designed to withstand centrifugation forces and provide a secure and leak-proof environment for the samples during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Thermal mixer, by Thermo Fisher Scientific (2 mentions)
Magnetic stirrer, by Thermo Fisher Scientific (2 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (2 mentions)
Toluidine blue solution, by Merck Group (1 mentions)
Dpx mountant, by Avantor (1 mentions)
Prism 7, by GraphPad (1 mentions)
Glycogen, by Thermo Fisher Scientific (1 mentions)
Rnase free disposable pellet pestle, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
6 protocols using 1.5 ml microcentrifuge tube
1
Purification and Elution of Frataxin Proteins
2
Purification of Recombinant Frataxin Proteins
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Supernatants with unbound proteins were removed and the beads re-suspended in bead wash buffer (1 mL). The beads were washed twice with bead wash buffer (1 mL) then transferred to clean 1.5 mL microcentrifuge tubes (ThermoFisher Scientific, Waltham, MA). After washing the beads with PBS (1 mL), hFXN-M and mFXN-M were eluted with 200 μL of elution buffer (100 mM acetic acid in 10% ACN) by shaking the beads in a thermal mixer (ThermoFisher Scientific, Waltham, MA) at 1000 rpm and RT for 1 h. Supernatants were then transferred to Sarstedt 2.0 mL LB microtubes and dried under a nitrogen flow using an N-Evap concentrator (Organomation, Berlin, MA).
3
Chondrogenic Differentiation of Adipose and Bone Marrow MSCs
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Three adipose-derived MSC's, three bone marrow-derived MSC's, all at passage 3 were tested. 2.5 x 10 5 MSC were aliquoted into 1.5ml polypropylene 1.5ml microcentrifuge tubes (Fischer Scientific, UK). These were then spun at 500g for 5 minutes to pellet the cells. MSC media was then aspirated and either fresh MSC media or chondrogenic media added. 5 tubes of each cell and media type were run. After 12 hours the cell pellet was gently detached from the bottom to become freefloating by pipetting. Media was then changed every 2-3 days for 21 days. RNA extraction was performed on 2 pellets in each experiment. The three remaining pellets were fixed in 4% formaldehyde and Toluidine blue staining performed. Pellets were embedded in paraffin wax blocks and sections cut using a microtome. Dewaxing was performed using xylene for 15 minutes, descending concentrations of ethanol (100, 95, 90, 70, and 0%) for 10 minutes each. The sections were rinsed in distilled water and submerged in 1% aqueous Toluidine blue solution (Sigma Aldrich) for 30 minutes at room temperature. The slides were then rinsed in 1 part distilled water and 4 parts 1% HCl in 70% ethanol for 5 seconds. The slides were then rinsed in distilled water, dried and mounted using DPX mountant (VWR, UK). 9
4
In Vitro Drug Release Kinetics
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Since both VAN and CFZ are hydrophilic drugs with negative log P values (−3.1 and −0.58, respectively), we evaluated the drug release kinetics of CFZ as a representative drug. In vitro drug release study was performed by a dialysis method modified in-house. This method used a minimum volume of formulation and release buffer, thus avoiding wastage of materials. Briefly, 200 μL of LCFZ was introduced to Slide-A-Lyzer™ Mini dialysis device, 3.5 kDa MWCO (Thermo Fisher Scientific, Waltham, MA, USA), which was plugged into a 1.5 mL microcentrifuge tube (Thermo Fisher Scientific, Waltham, MA, USA) containing 1 mL of PBS pH 7.4 as release buffer. The assembly was placed on a magnetic stirrer (Thermo Fisher Scientific, Waltham, MA, USA) at 350 rpm, 37 °C. Aliquots of 500 μL were removed from the release buffer at pre-determined time points up to 72 h. An equal volume of release buffer was replaced at every time point to maintain sink-conditions. The aliquots were analyzed by HPLC to quantify CFZ released in the buffer. The graph of % cumulative release versus time (hours) was plotted using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA) with n = 2.
5
In Vitro Drug Release Study of LVAN
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In vitro drug release study was performed using dialysis method modified in-house, as reported by us in [21 (link)]. Briefly, 500 μL of LVAN was introduced to Slide-A-Lyzer™ Mini dialysis device, 3.5 kDa MWCO (Thermo Fisher Scientific, Waltham, MA, USA), which was plugged into a 1.5 mL microcentrifuge tube (Thermo Fisher Scientific, Waltham, MA, USA) containing 1.5 mL of PBS pH 7.4 as release buffer. The assembly was placed in a water bath on a magnetic stirrer (Thermo Fisher Scientific, Waltham, MA, USA) at 350 rpm, 37 °C. Aliquots of 100 μL were removed from the release buffer over 72 h. An equal volume of release buffer was replaced at every time point to maintain sink conditions. The aliquots were analyzed by HPLC to quantify VAN released in the buffer. The graph of % cumulative release versus time (hours) was plotted (n = 2).
6
Spider RNA Extraction Protocol
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First, we selected one individual for each spider species, and dried the spider specimen using filter paper (Fig. 1a ). Second, we transferred the dried specimen into a new 1.5 ml microcentrifuge tube (Thermo Fisher Scientific, USA), and grinded the sample into a fine powder using RNase-Free Disposable Pellet Pestles (Thermo Fisher Scientific, USA) on liquid nitrogen. Third, we added 900 μl Trizol (Invitrogen, USA) and 8 μl glycogen (Thermo Fisher Scientific, USA) to maximize the amount of pure RNA (Fig. 1a ). Finally, we extracted total RNA from all selected individuals of 16 spider species following the Trizol manufacturer’s protocol (Fig. 1a ).
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