Zeba desalting spin columns 7 k mwco

The density of FBS was adjusted to 1.21 g/mL with sodium bromide. The solution was transferred to 11.5 mL polyallomer ultracrimp tubes and centrifuged for 20 h at 70,000× g in a Sorvall WX ultracentrifuge. The base of the tube was pierced with a butterfly needle and 1 mL aliquots collected. The cholesterol and total protein of the aliquots was measured using the Amplex™ Red Cholesterol Assay Kit (Life Technologies, Carlsbad, CA, USA) and the Pierce BCA Protein Assay Kit (ThermoFisher, Waltham, MA, USA), respectively, to determine which aliquots were deficient in lipoproteins (LPDS) and those enriched in lipoproteins (HLPS). Salt was removed from the serum by centrifugation with Zeba™ desalting spin columns 7 K MWCO (ThermoFisher) and serum was sterilized with a Millex 0.22 um syringe filter prior to storage at −20 °C.

We purified AGP from patient sera using a pH based precipitation method^{32 (link)} with some modification. This method enabled efficient isolation of AGP from high abundance blood proteins without the use of an antibody for affinity capture.
Serum samples were placed on ice and thawed immediately before use. 1.2 M perchloric acid was added to 20 μL of serum at a 1:1 (v/v) ratio, which can precipitate most of the impurity proteins while AGP remains in the liquid phase. The resulting 0.6 M perchloric acid concentration provided optimum recovery of AGP with minimal contamination by other proteins^{32 (link)}. Partial desialylation may occur at this concentration of perchloric acid, but Schultze et al. demonstrated that no sialic acid is released from AGP after short exposure to HClO₄ at low temperatures^{39 (link)}. We performed this procedure at low temperature (4 °C) and decreased the exposure time to HClO₄ to prevent this cleavage.
The mixture was vortexed for 20 s and then centrifuged at 3000 × g for 20 min at 4 °C to remove the precipitated proteins. The supernatant was pipetted, and transferred to another tube followed by the addition of 0.5 M NaOH solution to neutralize the acidic media. Desalting was achieved by using a 0.5 mL of Zeba desalting spin columns, 7 K MWCO (Thermo Scientific, Rockford, IL) and then the sample was dried in a SpeedVac concentrator (Thermo).

100 µM of Dylight® 488 was used to label TDP-43 LCD for fluorescent microscopy measurements. TDP 43 LCD (55 µM) in 1x PBS was added to the dye and incubated at room temperature for 30 min in darkness. The mixture was then desalted with the Zeba™ Desalting Spincolumns, 7 K MWCO (Thermo Fisher Scientific) to the reaction buffer (1x PBS, 10 mM MgCl₂, 0.05% Tween 20). Fluorescently labeled TDP-43 LCD was protected from light and stored at −80 °C.