Ring aggregates formed from rSslE were scraped from glass tubes and resuspended in 0.5 ml 100 mM citrate-phosphate buffer, pH 4.0, centrifuged at 15,000 × g and then the top 950 µl solution was carefully removed and discarded. This was followed by three rounds of addition of 950 µl 100 mM citrate-phosphate buffer at pH 4.0, centrifugation at 15,000 × g and the top 950 µl discarded. The final 50 µl sample was mixed with 1 × NuPAGE LDS Sample Buffer (ThermoFisher), 5% (v/v) β-mercaptoethanol and incubated at 100 °C for 5 min prior loading. This was run on a Criterion 4–20% SDS-PAGE gel (Bio-Rad), followed by transfer onto a PVDF membrane using the semi-dry Invitrogen Power–Blotter and Power–Blotter transfer blotting solution. The membrane was blocked in 1% (w/v) BSA, PBS-Tween for 1 h at room temperature followed by the addition of 1:2000 dilution mouse anti-His6 antibody (Sigma) in 0.5% (w/v/) BSA, PBS-Tween incubation buffer for 2 h. After five rounds of washing with incubation buffer, the membrane was incubated with 1:2000 anti-mouse HRP-conjugated antibody (Sigma) for 1 h, followed by five further washes and then treatment with ELC substrate (Peirce) before detection. Raw immunoblots are provided in Supplementary Fig. 23a .
Criterion 4 20 sds page gel
Manufactured by Bio-Rad
The Criterion 4–20% SDS-PAGE gel is a pre-cast polyacrylamide gel used for the separation and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel features a continuous 4–20% linear gradient of polyacrylamide, which allows for the effective separation of a wide range of protein molecular weights.
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4 protocols using criterion 4 20 sds page gel
1
Isolation and Analysis of His-tagged Proteins
2
Recombinant ChiA-CTD Binding to C1-INH
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Recombinant ChiA-CTD, ChiA-CTDD504A, ChiA-CTDH506A, ChiA-CTDE543M, ChiA-CTDH544A, ChiA-CTDN547A, ChiA-CTDQ583A, ChiA-CTDQ595A and ChiA-CTDQ617A were incubated for 5 min with 5 mM EDTA to remove any bound metal ions. They were then dialyzed extensively against PBS with 1 mM ZnCl2 and the concentrations adjusted to 50 μg/ml. 50 μl of ChiA sample was mixed with 50 μl of human C1-INH (1 mg/ml; Sigma) and incubated for 3 hr at 25°C. Reactions were stopped by adding 10 μl of 0.5 M EDTA. Samples were then run on a Criterion 4–20% SDS-PAGE gel (Bio-Rad) and visualized using Pro-Q Emerald 300 glycoprotein stain (Thermo Fisher Scientific).
3
Immunoblotting of E. coli surface proteins
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A lawn of wild-type E. coli W, BL21 (DE3), H10407 and E2348/69 strains or their derivatives were scraped from the surface of LB agar or YESCA agar plates after 96 h at 37 °C or 25 °C and transferred to 1 ml PBS and the volume adjusted to an OD600nm = 1.0. Formic acid was added to 70% (v/v) and then lyophilised overnight. Samples were resuspended in 200 µl 1× NuPAGE LDS loading buffer (ThermoFisher) and run on a Criterion 4–20% SDS-PAGE gel (Bio-Rad), followed by transfer onto a PVDF membrane using the semi-dry Invitrogen Power–Blotter and Power–Blotter transfer blotting solution. The membrane was blocked with 3% (w/v) BSA, PBS-Tween for 1 h at room temperature followed by the addition of 1:1000 dilution polyclonal primary anti-rSslE antibody (rabbit; Invitrogen) or 1:5000 dilution polyclonal anti-CsgA antibody (guineapig; Invitrogen) in 0.5% (w/v/) BSA, PBS-Tween incubation buffer for 1 h. After 3 rounds of washing with incubation buffer, the membrane was incubated with 1:2000 HRP-conjugated anti-rabbit or anti-guineapig antibody (Sigma), respectively, for 1 h, followed by three further washes and then treatment with ELC substrate (Peirce) before detection. Raw immunoblots are provided in Supplementary Fig. 23b, c .
4
SslE Protein Expression Analysis in E. coli
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E. coli W strains were grown on LB agar with 50 µg/ml kanamycin for Δssle mutants and 50 µg/ml kanamycin, 25 µg/ml chloramphenicol for Δssle::pCPC1 (sslE) and Δssle::pCPC2 (sslEΔM60). Single colonies were resuspended in 10 ml LB (with appropriate antibiotics) and incubated overnight at 37 °C with shaking (200 rpm). Cells were centrifuged to separate out the media (supernatant) and pellet (whole cells) and 3 µl of samples were resuspended in 17 µl of 1× NuPAGE LDS Sample Buffer (ThermoFisher), 5% (v/v) β-mercaptoethanol. Samples were run on a Criterion 4–20% SDS-PAGE gel (Bio-Rad), followed by transfer onto a PVDF membrane using the semi-dry Invitrogen Power–Blotter and Power–Blotter transfer blotting solution. The membrane was blocked with 1% (w/v) BSA, PBS-Tween for 1 h at room temperature, and then incubated overnight at 4 °C with polyclonal anti-rSslE antibody (rabbit; Invitrogen) or monoclonal anti-DsbA (mouse; Invitrogen), diluted 1:1000 using 0.5% (w/v) BSA, PBS-Tween incubation buffer. After three, 5 min washes with incubation buffer, membranes were incubated for 1 h at 37 °C with either anti-rabbit or anti-mouse secondary antibody conjugated to HRP (1:2000 dilution; Invitrogen) for 1 h at 25 °C and then treated with enhanced chemiluminescence substrate (ECL; Pierce) before detection.
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