P. edwinii colonies were scraped from their growth plates and resuspended in phosphate buffer saline before being pelleted. Supernatants were frozen and thawed to encourage precipitation of large particulate condiments, and centrifuged using a cellulose acetate Spin-X column (VWR). 20 ul of the supernatant was injected onto a Waters e2695 Separations Module equipped with a 2998 PDA Detector and run through a C18 column (XBridge, 3.5 um, 2.1 × 50 mm) housed at 40 °C at a flow rate of 0.5 ml min−1 for 20 mins. The mobile phase consisted of ddH20 + 0.04% (v/v) NH4OH with a gradient to 70% (v/v) acetonitrile + 0.04% (v/v) NH4OH with a constant background of 2% (v/v) methanol and compared against a prepared standard. The identify of PCA was confirmed using a quadrupole Time of Flight MS (Q-TOF, Xevo G2-XS, Waters) targeting a mass of 225.1 m/z.
Spin x column
Manufactured by Avantor
The Spin-X column is a specialized laboratory equipment designed for the efficient separation and purification of biomolecules. It features a centrifugal design that allows for the rapid and effective separation of samples, making it a valuable tool for various applications in the field of biochemistry and molecular biology.
Automatically generated - may contain errors
4 protocols using spin x column
1
Isolation and Identification of PCA
2
Immunostaining of hiPSCs and Derivatives
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hiPSCs and hiPSC-NPCs were fixed using 4% paraformaldehyde (PFA) and incubated at room temperature for fifteen minutes. hiPSC-neurons were fixed with 4% PFA added to cell culture medium at 50% for twenty-five minutes to maintain neuron attachment. After fixation, cells were washed with PBS, wrapped in parafilm and stored at 4°C prior to staining. PFA-fixed hiPSCs and hiPSC-derived cells were treated with blocking/permeabilizing buffer consisting of 3% BSA +0.3% Triton X-100 in PBS for 25 min (45 min for hiPSC-neurons) at room temperature. Primary antibodies were diluted in blocking/permeabilizing buffer and incubated on cells overnight at 4°C. After several washes with PBS, AlexaFluor secondary antibodies (ThermoFisher) were diluted at 1:500 in blocking/permeabilizing buffer and incubated for one hour at room temperature while protected from light. After several washes with PBS, DAPI was diluted in PBS and incubated for seven minutes at room temperature while protected from light. After incubation, cells were washed with PBS, protected from light, and stored at 4°C. To remove debris and improve imaging, all primary and secondary antibody dilutions and DAPI were filtered through a Spin-X column (VWR 29442-752) before being added to the cells.
3
Insoluble Tau Aggregate Analysis in hiPSC-Neurons
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For analysis of insoluble tau aggregates, hiPSC-neurons were fixed with 100% ice-cold methanol for fifteen minutes at −20°C. After fixation, cells were washed with PBS, wrapped in parafilm and stored at 4°C prior to staining. Fixed cells were treated with blocking buffer consisting of 3% BSA in PBS for 45 min at room temperature. Primary antibodies were diluted in blocking buffer before being added to the cells used and incubated overnight at 4°C. After several washes with PBS, AlexaFluor secondary antibodies (ThermoFisher) were diluted at 1:500 in blocking buffer and incubated for one hour at room temperature while protected from light. After several washes with PBS, DAPI was diluted in PBS and incubated for seven minutes at room temperature while protected from light. After incubation, cells were washed with PBS, protected from light, and stored at 4°C. To remove debris and improve imaging, all primary and secondary antibody dilutions and DAPI were filtered through a Spin-X column (VWR 29442-752) before being added to the cells.
4
Purification and Identification of PCA
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P. edwinii colonies were scraped from their growth plates and resuspended in phosphate buffer saline before being pelleted. Supernatants were frozen and thawed to encourage precipitation of large particulate condiments, and centrifuged using a cellulose acetate Spin-X column (VWR). 20 ul of the supernatant was injected onto a Waters e2695 Separations Module equipped with a 2998 PDA Detector and run through a C18 column (XBridge, 3.5 um, 2.1 x 50 mm) housed at 40 ºC at a flow rate of 0.5 ml min -1 for 20 mins. The mobile phase consisted of ddH20 + 0.04% (v/v) NH4OH with a gradient to 70% (v/v) acetonitrile + 0.04% (v/v) NH4OH with a constant background of 2% (v/v) methanol and compared against a prepared standard. The identify of PCA was confirmed using a quadrupole Time of Flight MS (Q-TOF, Xevo G2-XS, Waters) targeting a mass of 224.2 m/z.
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