Human serum albumin (10 mg, 149 nmol) was dissolved in 2 mL phosphate buffer (20 mM, pH 9.0) and added ATTO 647 N-hydroxysuccinimide ester (0.03 mg, 37.3 nmol) predissolved in anhydrous DMF. The reaction was stirred at room temperature for 16 h and purified using Sephadex G25M (GE Healthcare) size exclusion chromatography using MilliQ water as the mobile phase. The desalted solution was subsequently freeze dried to obtain ATTO 647-HSA in 92% yield.
Sephadex g 25 m
Manufactured by GE Healthcare
Sourced in Germany
Sephadex G-25 M is a size-exclusion chromatography medium used for desalting and buffer exchange of small molecules and proteins. It is a porous bead-formed gel, with a fractionation range suitable for separating molecules based on their size and molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Enzyme Research (3 mentions)
Penicillin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Millipore express plus, by Merck Group (1 mentions)
Stericup quick release, by Merck Group (1 mentions)
Cy5 nhs, by Lumiprobe (1 mentions)
Microcal peaq itc analysis software, by Malvern Panalytical (1 mentions)
Amicon ultra 4 centrifugal filter device, by Merck Group (1 mentions)
Microcal peaq itc, by Malvern Panalytical (1 mentions)
Alexa fluor 680 nhs ester, by Thermo Fisher Scientific (1 mentions)
Earle s salts, by Corning (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Amicon ultra 15 3 kda centrifugal filter unit, by Merck Group (1 mentions)
Earle s salt, by Merck Group (1 mentions)
Fetal calf serum (fcs), by GE Healthcare (1 mentions)
Amicon ultra 15 tube, by Merck Group (1 mentions)
12 protocols using sephadex g 25 m
1
ATTO 647-Labeled Human Serum Albumin
2
Expression and Purification of Recombinant Proteins
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Recombinant STA, RA, biotinylated IDR proteins, and LAF-fused Dronpa proteins were all prepared as previously described24 (link). Briefly, biotinylated IDR proteins were expressed in AVB101 (Avidity) cells, and other proteins were expressed in Escherichia coli BL21 (DE3). All proteins were purified by Ni-IDA columns (BioProgen, Daejeon, South Korea). Avidin proteins were produced as inclusion bodies, which were dissolved in 6 M Guanidine hydrochloride (GuHCl) and 50 mM Tris-HCl pH 8.0 for overnight at 4 °C. For refolding, denatured STA and RA proteins were diluted dropwise into PBS and filtered through a 0.22 μm membrane filter (Stericup® Quick Release, Millipore Express® PLUS), followed by overnight incubation at 4 °C before column purification. IDR-fused proteins were stored in a buffer containing 200 mM NaCl, 50 mM Tris pH 8.0, and 10% Glycerol. 1.0 LAF-Dronpa and 0.5 C LAF-Dronpa were labeled with NHS-Cy5 (Lumiprobe) by mixing proteins with a dye in a 1 : 0.5 protein/dye ratio. The mixed solutions were incubated for 40 min at 25 °C with shaking, and Cy5-labeled LAF-Dronpa was purified by a PD10 desalting column (Sephadex™ G-25 M, GE Healthcare). Protein concentrations were determined by Bradford assays and OD280 nm measurements with the Beer–Lambert equation.
3
Site-specific trastuzumab ADC synthesis
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To trastuzumab (5.0
μg/μL, 250 μg, 50 μL) in BBS buffer pH 8.0
was added TCEP (1 mM, 10.1 μL, 6 equiv) and the reaction was
incubated at 37 °C for 1 h 15 min. Linker3b in
DMSO (1 mM, 10.1 μL, 6 equiv) was added and the resulting solution
was mixed at 4 °C for 16 h (600 rpm). An iteration of incubation
with TCEP (1 mM, 5.0 μL, 3 equiv) at 4 °C for 1 h 15 and
mixing with linker3b (1 mM, 5.0 μL, 3 equiv) in
DMSO at 4 °C (600 rpm) for 22 h was done. The crude ADC was purified
by a PD-10 desalting column (Sephadex G-25 M, GE Healthcare) in PBS
Gibco pH 7.4 to afford the desired ADC4b (61%).
μg/μL, 250 μg, 50 μL) in BBS buffer pH 8.0
was added TCEP (1 mM, 10.1 μL, 6 equiv) and the reaction was
incubated at 37 °C for 1 h 15 min. Linker
DMSO (1 mM, 10.1 μL, 6 equiv) was added and the resulting solution
was mixed at 4 °C for 16 h (600 rpm). An iteration of incubation
with TCEP (1 mM, 5.0 μL, 3 equiv) at 4 °C for 1 h 15 and
mixing with linker
DMSO at 4 °C (600 rpm) for 22 h was done. The crude ADC was purified
by a PD-10 desalting column (Sephadex G-25 M, GE Healthcare) in PBS
Gibco pH 7.4 to afford the desired ADC
4
Conjugation of Trastuzumab with Linker 3a
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To trastuzumab (5.0
μg/μL, 250 μg, 50 μL) in PBS buffer pH 8.3
was added TCEP (1 mM, 13.5 μL, 8 equiv) and the reaction was
incubated at 37 °C for 2 h. Linker3a in DMF (1
mM, 12.7 μL, 7.5 equiv) was added under magnetic stirring and
the resulting solution was mixed at 20 °C for 1 h (600 rpm).
The crude ADC was purified by a PD-10 desalting column (Sephadex G-25
M, GE HealthCare) in PBS Gibco pH 7.4 to afford the desired ADC4a (75%).
μg/μL, 250 μg, 50 μL) in PBS buffer pH 8.3
was added TCEP (1 mM, 13.5 μL, 8 equiv) and the reaction was
incubated at 37 °C for 2 h. Linker
mM, 12.7 μL, 7.5 equiv) was added under magnetic stirring and
the resulting solution was mixed at 20 °C for 1 h (600 rpm).
The crude ADC was purified by a PD-10 desalting column (Sephadex G-25
M, GE HealthCare) in PBS Gibco pH 7.4 to afford the desired ADC
5
Menin-Compound Binding Affinity by ITC
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A solution of purified menin (1–615 with GST-tag cleaved as described above) was prepared in ITC buffer (50 mM sodium phosphate pH 7.5, 50 mM NaCl, 1 mM beta-mercaptoethanol) using a PD-10 desalting column (Sephadex G-25 M, GE Healthcare) and concentrated to a final concentration of 56 µM using an Amicon Ultra-4 centrifugal filter device (Millipore, Burlington, MA, USA). The compound was dissolved in 100% DMSO to a concentration of 10 mM and subsequently diluted to a final concentration of 5 µM in ITC Buffer. Isothermal Titration Calorimetry (ITC) was performed at 25 °C in a Microcal PEAQ-ITC (Malvern Panalytical). 5 µM compound was titrated with 56 μM menin (1–615) in ITC Buffer using injections of 2 μL within 4 s at 100-s intervals and a stirring speed of 1000 rpm. Reference Power was set to 6 μcal/s. Data were collected on high feedback mode. Analysis was performed with a single binding site model in the Microcal PEAQ-ITC Analysis Software (Malvern Panalytical).
6
Preparation of Fluorescent Fibrinogen
7
Neospora caninum Tachyzoites Isolation
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Neospora caninum tachyzoites (NcT) from strain Nc-1 (ATCC™ 50,843) were isolated from infected VERO cell cultures as previously mentioned, with slight modifications28 (link),31 (link),32 . Briefly, VERO cells infected with NcT were cultured at 37 °C and 5% CO2, in minimum essential medium with Earle’s salts (Corning, NY, Missouri, USA) supplemented with 2 mM L-glutamine, 200 units/mL penicillin and 200 μg/mL streptomycin (all from Sigma-Aldrich) and 10% FBS (Biowest), till 70% destruction of host cell monolayer. All the contents of the flask (the adherent cells collected using a cell scraper and the culture supernatants) were centrifuged at 1500×g for 20 min. The pellet was then passed through a 25G needle and washed three times in complete RPMI medium (all centrifugations were done at 1500×g for 20 min). The final pellet was resuspended in 3 mL of complete RPMI medium and passed through a PD-10 column filled with Sephadex G-25 M (GE Healthcare Life- Sciences, Freiburg, Germany). Freeze-killed NcT were prepared from suspensions of live tachyzoites (prepared as described above) resuspended in complete RPMI and kept at least one week frozen at − 80 °C, since others have previously shown that a 2 h freezing incubation at − 70 °C was enough to inactivate NcT33 (link).
8
Protein Refolding and Purification Protocol
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The purified protein sample was dissolved in deionized water containing 6 M GdmCl, 0.1 M Na2HPO4, 20 mM TCEP at pH 7.0, to obtain a concentration of 1.5 mM. The mixture was shaken overnight at 25 °C. Salts were removed with a PD-10 desalting column (Sephadex G-25 M, GE Healthcare). The obtained product was lyophilized and subsequently dissolved in 10 mM HCl and slowly added to degassed buffer containing 0.1 M Tris, 0.2 M KCl, 1 mM EDTA, 1 M GdmCl, 150 μM GSSG and 300 μM GSH at pH 8.7 with a final protein concentration of 30 μM. The refolding reaction was carried out at 37 °C for 24 h. The sample was concentrated with Amicon® Ultra-15 3 kDa Centrifugal Filter Unit (Merck, Darmstadt, Germany), followed by lyophilization. Refolded protein was further purified by RP-HPLC with a linear gradient starting with 10% to 80% B over 18 min at a flowrate of 15 mL min−1. Purity of fractions was analyzed by analytical RP-HPLC and ESI-MS (SI).
9
Isolation and Purification of N. caninum Tachyzoites
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N. caninum tachyzoites (NcT) (Nc-1, ATCC® 50843) were
propagated by serial passages in VERO cell cultures, maintained in Minimal
Essential Medium (MEM) containing Earle’s salts (Sigma, St. Louis,
MO, USA), supplemented with 10% fetal calf serum (PAA laboratories, Pasching,
Austria), L-Glutamine (2 mM), Penicillin (200 IU/ml) and
Streptomycin (200 g/ml) (all from Sigma), in a humidified atmosphere
with 5% CO2 at 37 °C. Free parasitic forms of
N. caninum were obtained as previously described4 (link).
Briefly, infected VERO cells were cultured until the host cell monolayer was 70%
destroyed. Free parasites and adherent cells were recovered using a cell scraper
and centrifuged at 1,500 × g for
15 min. The pellet was passed through a 25 G needle and then washed
three times in PBS by centrifugation at
1,500 × g for 15 min. The
resulting pellet was resuspended and passed through a PD-10 desalting column,
containing Sephadex™ G-25M (GE Healthcare, Freiburg, Germany).
Tachyzoites concentration was determined in a haemocytometer.
propagated by serial passages in VERO cell cultures, maintained in Minimal
Essential Medium (MEM) containing Earle’s salts (Sigma, St. Louis,
MO, USA), supplemented with 10% fetal calf serum (PAA laboratories, Pasching,
Austria), L-Glutamine (2 mM), Penicillin (200 IU/ml) and
Streptomycin (200 g/ml) (all from Sigma), in a humidified atmosphere
with 5% CO2 at 37 °C. Free parasitic forms of
N. caninum were obtained as previously described4 (link).
Briefly, infected VERO cells were cultured until the host cell monolayer was 70%
destroyed. Free parasites and adherent cells were recovered using a cell scraper
and centrifuged at 1,500 × g for
15 min. The pellet was passed through a 25 G needle and then washed
three times in PBS by centrifugation at
1,500 × g for 15 min. The
resulting pellet was resuspended and passed through a PD-10 desalting column,
containing Sephadex™ G-25M (GE Healthcare, Freiburg, Germany).
Tachyzoites concentration was determined in a haemocytometer.
10
Conjugation of trastuzumab with linker 3c
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To trastuzumab (5.0
μg/μL, 250 μg, 50 μL) in PBS buffer pH 8.3
was added TCEP (1 mM, 16.9 μL, 10 equiv) and the reaction was
incubated at 37 °C for 2 h. Linker3c in DMF (2
mM, 25.3 μL, 30 equiv) was added and the resulting solution
was mixed at 20 °C for 3 h (600 rpm). The crude ADC was purified
by a PD-10 desalting column (Sephadex G-25 M, GE Healthcare) in PBS
Gibco pH 7.4 to afford the desired ADC4c (72%).
μg/μL, 250 μg, 50 μL) in PBS buffer pH 8.3
was added TCEP (1 mM, 16.9 μL, 10 equiv) and the reaction was
incubated at 37 °C for 2 h. Linker
mM, 25.3 μL, 30 equiv) was added and the resulting solution
was mixed at 20 °C for 3 h (600 rpm). The crude ADC was purified
by a PD-10 desalting column (Sephadex G-25 M, GE Healthcare) in PBS
Gibco pH 7.4 to afford the desired ADC
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