Dylight-488 NHS-ester (Pierce) was dissolved in dimethyl formamide at 10 mg/mL. Recombinant human GDNF (Peprotech, Rocky Hill, NJ) was dissolved in 8 mM sodium phosphate buffer (pH 7.4). Dylight-488 was added to the solution for a final GDNF concentration of 10 μg/mL and a final Dylight-488 concentration of 50 ng/mL and incubated overnight at 2°C. The solution was then dialyzed using Slide-A-Lyzer MINI Dialysis Units (Thermo Scientific, Rockford, IL, 3500 MWCO) in 8 mM sodium phosphate buffer (pH 7.4) to remove unbound Dylight-488.
Slide a lyzer mini dialysis unit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Slide-A-Lyzer MINI Dialysis Units are lab equipment used for dialysis. They are designed for small-volume sample preparation and purification.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human gdnf, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
No weigh sulfo nhs biotin, by Thermo Fisher Scientific (3 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Yeast trna, by Merck Group (2 mentions)
Whatman fusion 5, by Cytiva (2 mentions)
Yeast trna, by Thermo Fisher Scientific (2 mentions)
Chitosan, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Myecl imager system, by Thermo Fisher Scientific (2 mentions)
Sk n be, by American Type Culture Collection (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent kit, by Thermo Fisher Scientific (1 mentions)
Ham s f 12 nutrient mix, by Thermo Fisher Scientific (1 mentions)
Sk n dz, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Chapso, by Merck Group (1 mentions)
Visking, by Serva Electrophoresis (1 mentions)
70 protocols using slide a lyzer mini dialysis unit
1
Protein Labeling with Dylight-488
2
Evaluating SPIO NPs and ASOs in Neuroblastoma
3
Cryo-EM Sample Preparation Protocol
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The complex was first dialyzed against 200 ml of intermediate buffer (20 mM Hepes, 200 mM KAc, 200 mM Na-Glu, 5 mM MgAc2, 0.5 mM TCEP, pH 7.5) for 2 h at 4 °C using Slide-A-Lyzer MINI Dialysis Units (7000MWCO) (Thermo Fischer Scientific). Then, a second dialysis was performed in 200 ml of the final cryo-EM buffer (20 mM Hepes, 100 mM KAc, 50 mM Na-glutamate, 5 mM MgAc2, 0.5 mM TCEP, pH 7.5) for 4 h at 4 °C. Finally, 8 mM CHAPSO (Sigma-Aldrich) was added to the dialyzed complex to prevent adsorption of the particles to the air/water interface47 (link). The sample was centrifuged for 2 h at 16,000 × g to remove potential aggregates.
4
Preparation of Hapten Conjugates
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Preparation of conjugates of hapten SPo was described in a previous article19 (link). Concerning SPm and SPh conjugates, compounds SPm-NHS and SPh-NHS were dissolved in DMF (50 mM) and coupled to BSA, OVA, and HRP in 100 mM phosphate buffer, pH 7.4, as described in the Supplementary Information file. A small aliquot of each conjugate solution was dialysed using Slide-A-Lyzer MINI dialysis units from Thermo Scientific. Hapten density of protein conjugates was determined by MALDI-TOF‒MS analysis in positive linear mode (1500 shots for each position) in a mass range of 10,000‒120,000 m/z, as described in the Supplementary Information file.
5
Protein Dialysis and Turbidity Measurement
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For ThT‐binding and turbidity measurement, specimens were dialyzed in 200 mmol/L acetate buffer containing 100 mmol/L NaCl and 1 mmol/L EDTA at pH 5.0 with Slide‐A‐Lyzer MINI Dialysis Units (Thermofisher Scientific). Afterwards, specimens from multiple columns were pooled and concentration was measured with a BioPhotometer (Eppendorf, Hamburg, Germany). Proteins were diluted with respective buffer to a final concentration of 0.244 mg/mL.
For time course turbidity assay, proteins were dialyzed against phosphate buffer solution (20 mmol/L sodium phosphate, 100 mmol/L NaCl, 1 mmol/L EDTA, pH 7.6) using a dialysis tube (VISKING, SERVA, Heidelberg, Germany) with a volume per length of 2 mL/cm and a molecular weight cut‐off of 12–14 kDa. After dialysis, the OD280 of each TTR‐variant was measured with a BioPhotometer (Eppendorf, Hamburg, Germany) and the gained protein concentrations were equaled by adding buffer (20 mmol/L sodium phosphate, 100 mmol/L NaCl, 1 mmol/L EDTA, pH 7.6), down to a protein concentration of 0.6 mg/mL.
For time course turbidity assay, proteins were dialyzed against phosphate buffer solution (20 mmol/L sodium phosphate, 100 mmol/L NaCl, 1 mmol/L EDTA, pH 7.6) using a dialysis tube (VISKING, SERVA, Heidelberg, Germany) with a volume per length of 2 mL/cm and a molecular weight cut‐off of 12–14 kDa. After dialysis, the OD280 of each TTR‐variant was measured with a BioPhotometer (Eppendorf, Hamburg, Germany) and the gained protein concentrations were equaled by adding buffer (20 mmol/L sodium phosphate, 100 mmol/L NaCl, 1 mmol/L EDTA, pH 7.6), down to a protein concentration of 0.6 mg/mL.
6
Purification of Amyloid Fibrils
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A 60 μl suspension of preformed mature fibrils (K3-TTR/FAM-E3-TTR/E3-TTR = 5/1/4) was mounted on 60 μl of a sucrose solution (62.5% w/v) in a centrifuge tube, and the resulting mixture was centrifuged at 43,000 ×g for 30 min at 20 °C. Forty microliters of the supernatant was removed and 80 μl of the residual solution was dialyzed with water (pH 2.0) using a 10 k MWCO dialyzer (Slide-A-Lyzer Mini Dialysis Units, Thermo Fisher Scientific, Waltham, MA, USA).
7
Fluorescent Labeling of GDNF Protein
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Example 15
Dylight-488 NHS-ester (Pierce) was dissolved in dimethyl formamide at 10 mg/mL. Recombinant human GDNF (Peprotech, Rocky Hill, N.J.) was dissolved in 8 mM sodium phosphate buffer (pH 7.4). Dylight-488 was added to the solution for a final GDNF concentration of 10 mg/mL and a final Dylight-488 concentration of 50 ng/mL and incubated overnight at 4° C. The solution was then dialyzed using Slide-A-Lyzer MINI Dialysis Units (Thermo Scientific, Rockford, Ill., 3500 MWCO) in 8 mM sodium phosphate buffer (pH 7.4) to remove unbound Dylight-488.
8
Fluorescent Heparin Labeling for Microspheres
9
Release Kinetics of CpG-Encapsulated Nanoparticles
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The release kinetics was determined in 10 mM sodium phosphate buffer (pH 7.0) and 10 mM acetate buffer (pH 5.0). The Slide-A-Lyzer MINI Dialysis units (10 kDa MWCO, Thermo Fisher Scientific) were loaded with 100 μL of CpG-encapsulated nanoparticles and placed in tanks containing aforementioned buffers at 37°C. After 15 min, 30 min, 1, 2, 4, 8, 24, 48, 72 and 96 hr, the nanoparticles remaining in dialysis units were collected and disrupted by 100% acetone. After acetone was evaporated, remaining CpG was analyzed by the reagent in Quant-iT™ RiboGreen® RNA Kit (Invitrogen).
10
Heparin Attachment to Microparticles
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Example 6
To confirm post-microparticle formation attachment, cysteinated heparin was labeled with Dylight-488 NHS-ester (Pierce). Cysteinated heparin (130 mg/mL) and Dylight-488 (560 mg/mL) in PBS was incubated overnight at room temperature. The labeled heparin solution was dialyzed using Slide-A-Lyzer MINI Dialysis Units (Thermo Scientific, Rockford, Ill., 3500 MWCO) in PBS (pH 7.4) to remove any unbound Dylight-488. The heparin solution was then used in the heparination post-microparticle formation protocol as described above. To determine heparin content, fluorescence of the suspended microparticle solution was measured using a plate reader in triplicate and compared to a standard curve of fluorescently labeled heparin in solution.
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