Fig 2: Protease Kinetics of Cathepsin L mediated PGRN Cleavage are Disrupted in Polypropylene Tubes. (A) 400 ng PGRN was incubated with 250 nM of Cathepsin L for 2.5, 5, 10 and 15 min in either polypropylene or LoBind tubes. (B,C) Quantification of the relative percentage of full-length PGRN and its approximately 75 kDa cleavage product after incubation with Cathepsin L for the aforementioned time points. Paired t-tests were performed (∗p < 0.05 in solution vs. adsorbed to tube for every condition, n = 3). All immunoblots are representative of three independent experiments.

Fig 3: PGRN Adsorption is Reduced in LoBind Tubes. (A) A dilution series of 250–25 nM PGRN was set up in LoBind tubes for 10-min incubations on ice (n = 3). (B) 100 nM PGRN was incubated for 10′, 1, 4, 8, 16, 24 or 48 h in LoBind tubes on ice (n = 3). (C) 100 nM PGRN was incubated in LoBind tubes for 10′ on ice, at room temperature (RT) or at 37°C (n = 3). At the end of the incubations, PGRN in solution as well as adsorbed to the tube was prepared for analysis. PGRN levels were measured by Western blot and membranes were immunoblotted with anti-PGRN antibody. All immunoblots are representative of three independent experiments.

Fig 4: Recombinant Progranulin Protein Adsorbs to Polypropylene Tubes. (A,B) 100 nM rhPGRN was serially transferred through tubes 1–5, which were stripped for adsorbed protein after a 10-min incubation in each tube. Tube 6 contains the protein remaining in solution at the end of five serial transfers. The control is 100 nM PGRN that underwent no transfers. Low binding pipet tips were used to minimize pipetting loss. One-Way ANOVA with Holm-Sidak’s Multiple Comparisons test was performed (****p < 0.0001, 1–6 vs. Control, n = 3). (C) A dilution series of 250–25 nM PGRN was set up in polypropylene tubes. After a 10-min incubation on ice, the PGRN in solution as well as adsorbed to every tube was prepared for analysis. Low binding pipet tips were used to minimize pipetting loss. (D) 100 nM PGRN was prepared in tubes 1–6. Polypropylene tips were used to aspirate and dispense the full volume of solution 0–5 times, respectively, into the same tube. The control is 100 nM PGRN subjected to no pipetting. PGRN levels were measured by Western blot and membranes were immunoblotted with anti-PGRN antibody. All immunoblots are representative of three independent experiments.

Fig 5: Time and Temperature Impact Adsorption of Recombinant Progranulin to Polypropylene Tubes. (A) 100 nM PGRN was incubated for 10′, 1, 4, 8, 16, 24, or 48 h in polypropylene tubes on ice. One-Way ANOVA with Dunnett’s Multiple Comparisons were performed (*p < 0.05, 1/4/8/16/24/48 h vs. 10′, n = 3). (B) 100 nM PGRN was incubated in polypropylene tubes for 10′ on ice, at room temperature (RT) or at 37°C (n = 3). At the end of the incubations, PGRN in solution as well as adsorbed to the tube was prepared for analysis. PGRN levels were measured by Western blot and membranes were immunoblotted with anti-PGRN antibody. All immunoblots are representative of three independent experiments.