InhA was labeled with the second-harmonic active amine reactive dye SHG1-SE (Biodesy, South San Francisco, CA). The labeling reaction was performed according to manufacturer’s instructions. Briefly, InhA was exchanged into cold 100 mM NaPO4, pH 7.5, 150 mM NaCl prior to labeling, using a 7K MWCO Zeba desalting column (ThermoFisher). The protein was then diluted into cold labeling buffer to a final concentration of 30 μM protein and a 2-fold molar excess of dye was added to the protein solution. The conjugation reaction was allowed to proceed for 10 min on ice followed by centrifugation for 20 min at 16.8K*g. The supernatant was then passed through a Zeba desalting column to remove free dye from the reaction and to exchange the protein into storage buffer (30 mM PIPES, pH 6.8, 150 mM NaCl, 8% glycerol). A Nano-Drop 2000 UV-Vis spectrophotometer (Thermo-Fisher) was used to determine the degree of labeling as 0.74 using the known extinction coefficients of the dye and the protein at the absorbance maxima of 410 nm and 280 nm, respectively.
7k mwco zeba desalting column
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The 7K MWCO Zeba desalting column is a size-exclusion chromatography product designed to remove small molecules, such as salts, from larger biomolecules like proteins. The column uses a proprietary resin to effectively separate the target analyte from contaminants based on differences in molecular weight.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
50k mwco amicon ultra centrifugal filter units, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Atto 647n, by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Thermo Fisher Scientific (1 mentions)
Dna 5k rna cze chip, by PerkinElmer (1 mentions)
Dbco sulfo nhs ester, by Jena Biosciences (1 mentions)
Affinipure goat anti mouse secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Hitrap mabselect sure, by GE Healthcare (1 mentions)
Hitrap protein g, by GE Healthcare (1 mentions)
Hitrap kappaselect, by GE Healthcare (1 mentions)
Captureselect iga affinity matrix, by Thermo Fisher Scientific (1 mentions)
Hitrap lambdafabselect, by GE Healthcare (1 mentions)
Hybridoma sfm, by Thermo Fisher Scientific (1 mentions)
Hitrap protein g, by Cytiva (1 mentions)
Hitrap mabselect sure, by Cytiva (1 mentions)
Rapigest, by Waters Corporation (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using 7k mwco zeba desalting column
1
Labeling InhA with SHG1-SE Fluorescent Dye
2
Labeling MLKL Proteins with SHG1-SE Dye
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The labeling reaction was performed according to manufacturer’s instructions. Briefly, His-MLKL-1 and His-MBP-MLKL-3 proteins were exchanged into 100mM NaH2PO4, pH 7.5, 150mM NaCl, 0.5mM TCEP prior to labeling with a 7K MWCO Zeba desalting column (Thermofisher). For the labeling reaction, a 5-fold molar excess of the second-harmonic-active dye SHG1-SE (available from Biodesy) was added to the protein solution and the reaction was allowed to proceed for 10 minutes at room temperature. The samples were then centrifuged for 20 minutes at 16.8K*g and the supernatant passed through a Zeba desalting column to remove free dye from the reaction. UV-Vis spectrophotometry was used to determine the degree of labeling.
3
Labeling of His-tagged PCNA and ClpB proteins
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His-tagged human PCNA protein was purchased from Sigma Aldrich (catalogue number SRP5117 ), at a concentration of 6.6 µM. The protein was specifically labeled via the histidine linker on the N-terminal side of the protein with the dye ATTO647N containing a Ni-NTA functional group which was also purchased from Sigma Aldrich (catalogue number 02175–250 UG-F). First, the protein was desalted to a labeling buffer (25 mM HEPES, 25 mM KCl, pH 7.8) using a 7 K MWCO Zeba desalting column (ThermoFischer, cat. 89882) and then reacted with dyes at a ratio of 1:4 for 2 hr at RT. The protein was then desalted from the excess of dyes using the same desalting column. The labeling efficiency was estimated using an absorption spectrometer (Nanodrop 2000, ThermoFischer) confirming ~100% labeling efficiency. SDS-page and native gel indicated that the protein is indeed assembled and labeled. N-terminal His-tagged ClpB protein, mutated at residue 428 from alanine to cysteine, was purified as described in a previous publication (Mazal et al., 2019 (link)). The labeling of the single cysteine was done following the same procedure as describe above with ATTO647N maleimide as a specific labeling agent of the cysteine. Absorption spectroscopy, SDS-page and native gel indicate complete assembly of the protein, see previous publication (Mazal et al., 2019 (link)).
4
Quantitative Analysis of mAb Charge Variants
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The relative mAb charge variant distribution was measured with the LabChip® GXII HT Touch™ (Perkin Elmer, CLS138160), the DNA 5K/RNA/CZE Chip (Perkin Elmer, 760435), and the Protein Charge Variant Reagent Kit (Perkin Elmer, CLS760670), according to the manufactures’ protocol. Briefly, purified IgG1 was diluted to 3.3 mg/ml in Tris/acetate buffer and then 80 μl was desalted with a 75 µl 7 K MWCO Zeba desalting column (Thermo Fisher Scientific, 89878). The concentration of each sample was measured using its A280 and then for each labeling reaction, adjusted to 25 μl of IgG1 at 2 mg/ml concentration with water. Labeling was performed as specified, using N,N‐dimethylformamide (Acros Organics, 61094‐1000) in addition to the components provided in the charge variant labeling kit. Care was taken to mix each sample in‐between the addition of each reagent. The DNA 5K/RNA/CZE Chip was prepared with pH 7.2 reagents and samples were analyzed with a 90 s analysis time. All samples were analyzed in triplicate. Peak areas were determined using Empower 3 FR5, similar to the analysis of µCE‐SDS described above.
5
Antibody Conjugation with Oligonucleotides
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Anchor oligonucleotides (Supplementary Table 1 ) were conjugated to secondary antibodies for immunolabeling of cardiac samples. Lyophilized oligonucleotides were resuspended in PBS (pH 7.4) to 100 µM and kept at −20 °C for long term storage until required for conjugation. AffiniPure Goat Anti-Mouse secondary antibodies (affinity purified, #115-005-003, Jackson ImmunoResearch, PA) were conjugated using click-chemistry as described by Schnitzbauer et al.2 (link) Briefly, the antibody was incubated with 10-fold molar excess DBCO-sulfo-NHS-ester (Jenabioscience, Germany) for 45 min. The reaction was quenched with 80 mM Tris-HCl (pH 8.0) for 10 min and then desalted using 7 K MWCO Zeba desalting columns (Thermo Fisher). A 10-fold molar excess of the azide modified oligonucleotide was then incubated with the DBCO-antibody mixture overnight at 4 °C. Subsequently the antibody was purified using 100 K Amicon spin columns (Sigma). The absorbance of the oligonucleotide-conjugated fluorophores (Cy3 or Cy5) was recorded with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham) and used to quantify the degree of labeling for each conjugation, typically achieving >1–3 oligonucleotides per antibody.
6
Hybridoma Cloning and Antibody Purification
7
Generating Humanized Monoclonal Antibodies
8
Muscle Redox Proteomics Sample Prep
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Sample preparation was performed as previously described [21 (link)], briefly each muscle (N = 5 for soleus tissue from adult and old mice and quadriceps tissue from old mice, N = 6 for quadriceps tissue from adult mice) was dissected and immediately placed in d0 NEM blocking buffer to prevent Cys oxidation. Tissues were homogenised in ice cold buffer (25 mM Ammonium bicarbonate containing 25 mM NEM and 0.1% Rapigest (Waters, Manchester, UK) pH 8) using a hand homogeniser. Samples were passed through a Zeba desalting column 7K MWCO (Thermo Scientific, Hempstead, UK) to remove excess d0 NEM and protein concentrations were calculated using the Bradford (BioRad, Hertfordshire, UK) method using BSA as a standard. 100 μg of protein was aliquoted and reversibly oxidised Cys residues were reduced using tris (2-carboxyethyl) phosphine (TCEP) at a final concentration of 10 mM, newly reduced Cys residues were subsequently labelled with d5 NEM final concentration 20 mM. Protein extracts were digested overnight at 37 °C with trypsin, Rapigest was precipitated by addition of trifluoroacetic acid (TFA) and subsequent centrifugation before analysis by MS.
9
Fab Fragment Preparation and Binding Kinetics
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Fab fragments were prepared using the Pierce Fab Preparation kit (Thermo Fisher Scientific, 44985) following the manufacturer’s protocol with slight modifications. Briefly, 250-500 μg of each cross-reactive mAb was digested using immobilized papain for 3 h at 37oC. The resulting digest was applied to Protein G Hi-Trap spin columns (Cytiva) to purify Fabs from Fc fragments and undigested mAbs. Residual reducing agent was removed using a Zeba Desalting Column 7K MWCO (ThermoScientific, 89882). Protein concentrations were determined using A280 measurements and Fab digests were confirmed using reducing and non-reducing SDS-PAGE.
For analysis of antibody binding kinetics, Fabs were coupled to an HC30M chip (0.56 μg/mL) and a three-fold dilution series of SARS-CoV-2 S2 subunit or spike protein was injected in ascending concentration without regeneration. A 10 min association and 30 min dissociation time were used. Association (ka) and dissociation rates (kd), as well as dissociation constants (KD) were calculated using the Kinetics Software (Carterra).
For analysis of antibody binding kinetics, Fabs were coupled to an HC30M chip (0.56 μg/mL) and a three-fold dilution series of SARS-CoV-2 S2 subunit or spike protein was injected in ascending concentration without regeneration. A 10 min association and 30 min dissociation time were used. Association (ka) and dissociation rates (kd), as well as dissociation constants (KD) were calculated using the Kinetics Software (Carterra).
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