Amicon ultrafiltration devices (10K cut off) were from Millipore. PD-SpinTrap G-25 columns were from GE Healthcare. Slide-A-Lyzer MINI Dialysis devices (20K cut off) were from Thermo. 96-well plates were from Falcon. Recombinant hamster vimentin was from Cytoskeleton, Inc. Recombinant human vimentin was from Biomedal (Sevilla, Spain). Other reagents were of the highest quality from Sigma.
Amicon ultrafiltration device
Manufactured by Merck Group
Sourced in United States
Amicon ultrafiltration devices are laboratory equipment used for the separation and concentration of macromolecules, such as proteins, enzymes, and nucleic acids, from complex mixtures. These devices employ a semi-permeable membrane to selectively allow the passage of smaller molecules while retaining the larger target molecules.
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Lab products found in correlation
Vacuum concentrator, by Eppendorf (2 mentions)
Captureselect c tagxl, by Thermo Fisher Scientific (2 mentions)
Fectopro, by Polyplus Transfection (2 mentions)
Mabselect sure, by GE Healthcare (2 mentions)
96 well plate, by BD (1 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (1 mentions)
Pd spintrap g 25 column, by GE Healthcare (1 mentions)
Spintrap, by Cytiva (1 mentions)
Vitrobot mark 4 plunging system, by Thermo Fisher Scientific (1 mentions)
Hiprep, by Cytiva (1 mentions)
Starfect high efficiency transfection reagent, by GenStar (1 mentions)
Histrap excel column, by Cytiva (1 mentions)
Brij 35, by Merck Group (1 mentions)
3100 offgel fractionator, by Agilent Technologies (1 mentions)
Oasis hlb extraction cartridge, by Waters Corporation (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Superdex 75 10 300 gl gel filtration column, by GE Healthcare (1 mentions)
Hitrap q hp column, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Histrap ff column, by GE Healthcare (1 mentions)
19 protocols using amicon ultrafiltration device
1
Vimentin Purification and Characterization
2
Cryo-EM sample preparation of IstA-DNA complex
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To obtain the IstA/right-TIR complex sample to perform cryo-EM experiments, IstA was mixed in a 1:1.5 (protein:DNA) molar ratio with the right TIR (Supplementary Table 1 ), containing a 5-nt 5’ overhang, and dialyzed overnight at 4 °C against 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 MgCl2, 5 % glycerol and 1 mM DTT. The sample was loaded onto a HiPrep™ Sephacryl S-200 16/60 HR column (Cytiva) equilibrated in dialysis buffer, at room temperature. The fractions corresponding to the peak containing IstA-DNA complex (~42 ml) were pooled and concentrated to 2.45 mg/ml (51.5 µM) using Amicon ultrafiltration devices (10 kDa cutoff; Millipore), flash-frozen with liquid nitrogen and stored at −80 °C.
For the cryo-EM experiments, the sample was diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.005% Tween-20 to improve the particle distribution and angular coverage. 4 μl of the IstA-DNA complex (2.5 µM) was applied to glow-discharged C-Flat holey grids (CF-1.2/1.3; 400 mesh), blotted for 2 s (blot force 0) and frozen in liquid ethane using a Vitrobot Mark IV plunging system (ThermoFisher Scientific).
For the cryo-EM experiments, the sample was diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.005% Tween-20 to improve the particle distribution and angular coverage. 4 μl of the IstA-DNA complex (2.5 µM) was applied to glow-discharged C-Flat holey grids (CF-1.2/1.3; 400 mesh), blotted for 2 s (blot force 0) and frozen in liquid ethane using a Vitrobot Mark IV plunging system (ThermoFisher Scientific).
3
Neoglycoconjugate-CRM197 Vaccine Preparation
4
Peptide Extraction from Kefir Mixtures
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Samples stored at −20 °C were thawed for peptide extraction from complex kefir mixtures. Kefir samples and non-fermented milk (control) were first filtered using Amicon ultrafiltration devices (Millipore, Bedford, MA, USA) with a membrane cut-off of 10 kDa. Four milliliters of each sample was loaded into the devices and centrifuged at 5000× g and 4 °C until the whole volume passed throughout the membrane. The peptides present in the flow-through membrane filtrates were further cleaned and concentrated using 1 cc-Oasis HLB extraction cartridges (Waters, Milford, MA, USA) according to manufacturer’s instructions and modified by our research group for peptide cleaning and concentration of samples from bacteria [17 (link),22 (link)]. Briefly, after conditioning the extraction cartridges with 80% ACN followed by 0.1% formic acid solution, 0.5 mL samples were loaded, and peptides were eluted with increasing concentrations (10, 20, and 50%) of ACN in 0.1% formic acid. Peptide fractions were completely dried using a vacuum concentrator (Eppendorf, Hamburg, Germany), and kept at −20 °C until further analysis.
5
Purification of Recombinant RBD Protein
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Expi293F cells were diluted to 1.5 × 106 cells/mL before transfection. Next, 50 μg of RBD constructs were transfected into 50 mL suspension Expi293F with StarFect high-efficiency transfection reagent (Genstar, Beijing, China). Following 4 days of culturing by shaking, media were collected and centrifuged to harvest the supernatant and cleared of debris by 0.45-μm filtration before loading onto a 1 mL HisTrap excel column (Cytiva). The column was washed for 5 column volumes (CVs) with wash buffer (0.2 mol/L PBS, pH = 8.0), and 5 CVs of wash buffer supplemented with 20 mmol/L imidazole to equilibrate the column. Next, samples were loaded in equilibration buffer (0.2 mol/L PBS, pH = 8.0, with 20 mmol/L imidazole), after that, protein was eluted with elution buffer (0.2 mol/L PBS, pH = 8.0, 500 mmol/L imidazole). Elution fractions containing the protein were collected for SDS-PAGE validation. Elution buffer was then changed with PBST (0.2 mol/L, 0.05% tween-20, pH = 8.0) for desalting and concentration using Amicon ultrafiltration devices (Millipore, 10 kDa). The protein concentrations of purified samples were determined based on BSA standard curve protein quantification method using BCA Protein Content Assay Kit (Beyotime, Shanghai, China). Protein samples were flash-frozen in liquid nitrogen and stored at −80 °C.
6
Protein Concentration via Ultrafiltration
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Whenever necessary, proteins were concentrated using Amicon ultrafiltration devices (Ultra À15; 3000 Da cut-off; Millipore) using centrifugation (3,000 x g, 4 C; Sigma 3-16KL Benchtop Centrifuge; 1180).
7
OFFGEL Fractionation and Purification
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OFFGEL fractionation was performed at the Scientific Park (Madrid, Spain) using a 3100 OFFGEL Fractionator (Agilent Technologies, Santa Clara, CA, USA). Analysis conditions were as follows: 24-well setup, IPG gel strip pH 6-9 linear (GE Healthcare, Little Chalfont, UK), nondenaturing conditions (5% glycerol, 1% IPG buffer pH 3-11). Running conditions were as follows: 20°C, until 64 kVh limited by 50 μA or 4500 V. To eliminate any interference from the OFFGEL Fractionator, OFFGEL fractions were filtrated through 100 kDa cutoff Amicon Ultra Filtration devices (Millipore). Filtrate containing PSA was then concentrated using a Microcon YM 10 kDa concentration device (Millipore) previously passivated with 5% Brij35® (Sigma-Aldrich, Steinheim, Germany). Concentrated OFFGEL fraction samples were analyzed by CZE and 2DE, as described in the next section.
8
Peptide Extraction from Kefir Samples
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Samples kept at -20 ºC were thawed for peptide extraction from complex kefir mixtures.
Peptides present in kefir samples and raw milk (control) were separated from non-totally digested proteins and large polypeptides using Amicon ultrafiltration devices (Millipore) with a membrane cut-off of 10 kDa. Four mL of each sample was loaded into the devices and centrifuged at 5,000 g until the whole volume passed throughout the membrane. The peptides contained in the flow-through membrane filtrates were further cleaned and concentrated using Oasis HLB extraction cartridges (Waters, Milford, MA, USA) according to manufacturer's instructions and modified by our research group for peptide cleaning and concentration of samples from bacteria (Rodriguez-Ortega, 2018) . Briefly, after conditioning the extraction cartridges with 80% ACN and then with 0.1% formic acid solution, 0.5 mL samples were loaded, and peptides were eluted with increasing concentrations (10, 20, and 50%) of ACN in 0.1% formic acid. Peptide fractions were totally dried with a vacuum concentrator (Eppendorf, Hamburg, Germany), resuspended in 100 µL of 2% ACN/0.1% formic acid and kept at -20 °C until further analysis.
Peptides present in kefir samples and raw milk (control) were separated from non-totally digested proteins and large polypeptides using Amicon ultrafiltration devices (Millipore) with a membrane cut-off of 10 kDa. Four mL of each sample was loaded into the devices and centrifuged at 5,000 g until the whole volume passed throughout the membrane. The peptides contained in the flow-through membrane filtrates were further cleaned and concentrated using Oasis HLB extraction cartridges (Waters, Milford, MA, USA) according to manufacturer's instructions and modified by our research group for peptide cleaning and concentration of samples from bacteria (Rodriguez-Ortega, 2018) . Briefly, after conditioning the extraction cartridges with 80% ACN and then with 0.1% formic acid solution, 0.5 mL samples were loaded, and peptides were eluted with increasing concentrations (10, 20, and 50%) of ACN in 0.1% formic acid. Peptide fractions were totally dried with a vacuum concentrator (Eppendorf, Hamburg, Germany), resuspended in 100 µL of 2% ACN/0.1% formic acid and kept at -20 °C until further analysis.
9
Purification of MbRIP1 Protein
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The purification of MbRIP1 was carried out using the protocol reported earlier [13] (link). The samples of purified protein were concentrated using Amicon ultrafiltration device (Millipore Corporation, Bedford, USA) with a membrane having a molecular weight cut off of 3 kDa.
10
Purification of Sunflower Allergen Hel a 6
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All the chromatographic procedures were performed using an ÄKTAprime plus (GE Healthcare Life Sciences) FPLC system14 (link). The crude pollen extract (5 mg/ml) was fractionated in HiTrap Q HP column (GE Healthcare Life Sciences) equilibrated with the same buffer followed by elution of column-bound proteins using a linear gradient of 0.1–1 M NaCl. Eluted fractions containing the desired protein at a higher level of purity were pooled, dialyzed against 20 mM phosphate buffer (pH 7.4), and then concentrated in < 10 kDa cut-off Amicon Ultrafiltration device (Millipore). About 2 mg of concentrated protein was loaded onto a Superdex 75 10/300 GL gel filtration column (GE Healthcare Life Sciences)15 (link). Eluted fractions were screened by IgE-western blot using serum of 4 sunflower-sensitized patients to check for the presence of Hel a 6. The fractions containing Hel a 6 at > 90% purity were pooled, dialyzed against 1 M ammonium bicarbonate buffer (pH 8.5), and lyophilized.
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