After incubating the enzyme with the test chemical, 10-mM dithiothreitol and × 4 Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture. The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19 (link),20 (link)]. The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures’ protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
4 laemmli sample loading buffer
Manufactured by Bio-Rad
Sourced in United States
× 4 Laemmli sample loading buffer is a concentrated solution used to prepare protein samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. It contains the necessary components to denature and reduce proteins, allowing for their separation based on molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Flamingo stain, by Bio-Rad (2 mentions)
Las 1000plus, by Fujifilm (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Precision plus protein dual color standard, by Bio-Rad (2 mentions)
Typhoon 9400 variable mode imager, by GE Healthcare (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Thpta, by Merck Group (1 mentions)
Cuso4, by Merck Group (1 mentions)
3 protocols using 4 laemmli sample loading buffer
1
Analysis of quinone-modified proteins
2
Covalent Probe Labeling of Mpro
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Recombinant Mpro was incubated with probes Alk-4d or Alk-4i at indicated concentrations
at 30 °C for 1 h or with the incubation time indicated for the
time-course experiment. Next, click chemistry was performed at a final
concentration of 25 μM TAMRA-azide, 1 mM tris(2-carboxyethyl)phosphine
(TCEP, Thermo Scientific), 100 μM Tris-hydroxypropyltriazolylmethylamine
(THPTA, Sigma-Aldrich), and 1 mM CuSO4 (Sigma-Aldrich)
in a total volume of 21 μL. The reactions were carried out at
room temperature for 1 h in the dark. Click reactions were terminated
by the addition of 7 μL of 4× Laemmli sample loading buffer
(Bio-Rad), boiled for 7 min, and resolved onto 4–20% sodium
dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel
(Bio-Rad). Fluorescence was visualized at 532 nm for excitation and
at 600 nm for emission on a Typhoon 9400 Variable Mode Imager (GE
Healthcare), and images were displayed as grayscale. After fluorescence
scanning, protein loadings were visualized by silver staining using
the Thermo Scientific Pierce Silver Stain Kit. Competition labeling
involved a cotreatment of recombinant Mpro (1 μg)
with a competitor (nirmatrelvir,4d , or 4i ) and a clickable probe (Alk-4d or Alk-4i ) at indicated concentrations at 30 °C for 1 h. All subsequent
steps on click reaction and gel imaging were the same as those above.
at 30 °C for 1 h or with the incubation time indicated for the
time-course experiment. Next, click chemistry was performed at a final
concentration of 25 μM TAMRA-azide, 1 mM tris(2-carboxyethyl)phosphine
(TCEP, Thermo Scientific), 100 μM Tris-hydroxypropyltriazolylmethylamine
(THPTA, Sigma-Aldrich), and 1 mM CuSO4 (Sigma-Aldrich)
in a total volume of 21 μL. The reactions were carried out at
room temperature for 1 h in the dark. Click reactions were terminated
by the addition of 7 μL of 4× Laemmli sample loading buffer
(Bio-Rad), boiled for 7 min, and resolved onto 4–20% sodium
dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel
(Bio-Rad). Fluorescence was visualized at 532 nm for excitation and
at 600 nm for emission on a Typhoon 9400 Variable Mode Imager (GE
Healthcare), and images were displayed as grayscale. After fluorescence
scanning, protein loadings were visualized by silver staining using
the Thermo Scientific Pierce Silver Stain Kit. Competition labeling
involved a cotreatment of recombinant Mpro (1 μg)
with a competitor (nirmatrelvir,
steps on click reaction and gel imaging were the same as those above.
3
Quinone Detection in Protein Samples
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After incubating the enzyme with the test chemical, 10-mM dithiothreitol and ×4
Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture.
The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19, 20] (link). The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures' protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture.
The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19, 20] (link). The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures' protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
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