The hybridoma 139H2 was loaded on a 4–12% Bis-Tris precast gel (Bio-Rad) in non-reducing conditions and run at 120 V in 3-MOPS buffer (Bio-Rad). Bands were visualized with Imperial Protein Stain (Thermo Fisher Scientific), and the size of the fragments was evaluated by running a protein standard ladder (Bio-Rad). The IgG bands were cut and reduced by 10 mM Tris(2-carboxyethyl)phosphine at 37°C, followed by alkylation in 40 mM IAA at RT in the dark. The gel bands were digested by chymotrypsin and thermolysin at 37°C overnight in 50 mM ammonium bicarbonate buffer. The peptides were extracted with two-step incubation at RT in 50% ACN and 0.01% TFA, and then 100% ACN, respectively.
Protein standard ladder
Manufactured by Bio-Rad
Protein standard ladder is a tool used in protein electrophoresis. It provides a set of known molecular weight proteins, which serve as markers to determine the molecular weights of unknown protein samples.
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Lab products found in correlation
4 protocols using protein standard ladder
1
Protein Analysis by SDS-PAGE and Mass Spectrometry
2
Purification and Analysis of Fab-SOSIP Complex
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Complex production was achieved by adding 15x Molar excess monoclonal Fab to 20ug trimer. This was then incubated overnight at room temperature. Fab-trimer complex was fixed by addition of glutaraldehyde to a final concentration of 5mM and then incubated for 10min at room temperature. Excess glutaraldehyde was quenched by adding sufficient 1M Tris and incubated for 10min at room temperature. Complex was purified through SEC by loading on a Superose 6 increase 10/300 column using a 100 μL loop, and run at 0.5 mL/min using an Ӓkta Pure system (GE Healthcare). Fractions were collected and complex peak pooled and concentrated by 10kDA cutoff centrifugal filtration (Amicon/EMD Millipore). Fab-SOSIP complex formation and quality was further accessed by SDS PAGE gel electrophoresis, where ~1 μg complex/lane was loaded on a 4%–15% TGX Stain free gel (BioRad) in both reducing and non-reducing conditions and run at 200 V in Tris/Glycine/SDS buffer. Bands were visualized using Gel Doc EZ imager (BioRad), and the size of the fragments assessed by a protein standard ladder (BioRad).
3
Fab Fragment Characterization
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Fab (3 μg/lane) was loaded on
a 4%–12% Bis-Tris precast gel (Biorad) in nonreducing conditions
and run at 120 V in a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer
(Biorad). Bands were visualized with Imperial Protein Stain (Thermo
Fisher Scientific), and the size of the fragments was evaluated by
running a protein standard ladder (Biorad). The Fab bands were cut
and reduced by 10 mM TCEP at 37 °C and then alkylated in 40 mM
IAA at RT in the dark, followed by alkylation in 40 mM IAA at RT in
the dark. The Fab bands were digested by trypsin, chymotrypsin, thermolysin,
and alpha lytic protease at 37 °C overnight in a 50 mM ammonium
bicarbonate buffer. The peptides were extracted with two steps of
incubation at RT in 50% ACN and 0.01% TFA, and then 100% ACN, respectively.
The peptides were dried in speed-vac. To obtain the sequence of the
glycosylated Fab, the N-linked glycan was removed by PNGaseF at 37
°C overnight and then in gel digested as described above.
a 4%–12% Bis-Tris precast gel (Biorad) in nonreducing conditions
and run at 120 V in a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer
(Biorad). Bands were visualized with Imperial Protein Stain (Thermo
Fisher Scientific), and the size of the fragments was evaluated by
running a protein standard ladder (Biorad). The Fab bands were cut
and reduced by 10 mM TCEP at 37 °C and then alkylated in 40 mM
IAA at RT in the dark, followed by alkylation in 40 mM IAA at RT in
the dark. The Fab bands were digested by trypsin, chymotrypsin, thermolysin,
and alpha lytic protease at 37 °C overnight in a 50 mM ammonium
bicarbonate buffer. The peptides were extracted with two steps of
incubation at RT in 50% ACN and 0.01% TFA, and then 100% ACN, respectively.
The peptides were dried in speed-vac. To obtain the sequence of the
glycosylated Fab, the N-linked glycan was removed by PNGaseF at 37
°C overnight and then in gel digested as described above.
4
Purification and Analysis of Fab-SOSIP Complex
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