The equivalent of 8 × 107 cells was resuspended in 0.5 ml of 1X Laemmli buffer, incubated at room temperature for 30 min and then cold-centrifuged for 15 min at 16,000 × g. The supernatant was separated, supplemented with 10 mM DTT and boiled for 5 min. After a second centrifugation, the supernatant was loaded and run on denaturing SDS-PAGE gels (4%/10%), followed by immunoblotting in standard conditions (Supplementary Fig. 5 ). Blots were probed with anti-CWP1 antibody (dilution 1:1,000; product no. A300 TR-R-20x, Waterborne Inc.) followed by anti-mouse horseradish peroxidase-coupled antibody (dilution 1:5000; product no. NB7539, Novus Biologicals) and developed using a chemiluminescent substrate (Westernbright; Thermofisher Scientific). Gels for loading controls were stained with Instant Blue Coomassie stain (Expedeon).
Instantblue coomassie stain
Manufactured by Abcam
Sourced in United Kingdom, United States
InstantBlue Coomassie stain is a ready-to-use protein stain solution. It is used to detect and quantify proteins in polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Xcell surelock system, by Thermo Fisher Scientific (4 mentions)
Storage phosphor screen, by GE Healthcare (3 mentions)
Mops buffer, by Thermo Fisher Scientific (3 mentions)
Typhoon fla 7000 imager, by GE Healthcare (3 mentions)
Gelair dryer, by Bio-Rad (3 mentions)
Gel drying solution, by Bio-Rad (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Chemidoc xrs imager, by Bio-Rad (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Novex tris glycine sds sample buffer, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Image lab version 5, by Bio-Rad (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Sigmafast bcip nbt tablet, by Merck Group (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting system, by GE Healthcare (1 mentions)
A5588, by Merck Group (1 mentions)
Dataanalysis 4, by Bruker (1 mentions)
Maxis 4g etd, by Bruker (1 mentions)
41 protocols using instantblue coomassie stain
1
Western Blot Analysis of CWP1 Protein
2
Quantifying Colicins and Immunity Proteins
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Radioactive 14C‐Leu was used in the CFPS reaction at a final concentration of 10 μM. Total and soluble fractions of each reaction were heat‐denatured and reduced by dithiothreitol and electrophoresed on 4%–12% NuPAGE sodium dodecyl sulfate‐polyacrylamide gels in MOPS buffer (Invitrogen). The gels were stained with InstantBlue Coomassie stain (Expedeon) for 1 h, destained overnight in dH2O, soaked in Gel Drying Solution (Bio‐Rad) for 30 min, fixed with cellophane films, dried overnight in a GelAir Dryer (Bio‐Rad), and exposed for 72 h on Storage Phosphor Screens (GE Healthcare Biosciences). The autoradiograms were scanned with a Typhoon FLA7000 Imager (GE Healthcare Biosciences). To determine the proportion of colicins to immunity proteins, we followed Davarinejad's protocol in the ImageJ software.22 Briefly, the band intensity determined by ImageJ was used to calculate the mass fraction. The molar fraction was calculated as the mass fraction and molar mass. The average molecular weight and average number of leucines were calculated based on the molar fraction of each protein (Table S1 ). These average values were used separately to quantify colicins and immunity proteins in each radioactive sample measured with 14C‐leucine radioactive scintillation counting (Table 2 ).
3
Flagella Isolation and SDS-PAGE Analysis
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The same MB liquid cultures were used as for RT-qPCR. Cells in 5 mL aliquots were pelleted by centrifugation at 3000 ×g for 10 min. The culture with the lowest OD600 was resuspended in 200 μL phosphate buffered saline by vigorous vortexing and the other cultures were resuspended in a volume to give an equal cell density. Flagella were depolymerised at 65 °C for 5 min in a water bath and the cells were removed by centrifugation at 17,000 ×g for 10 min. 25 μL of each supernatant was mixed with an equal volume of 2× Novex Tris-Glycine SDS Sample Buffer (Invitrogen, Inchinnan, U.K.) and 40 μL of the mixture was run on an 8% Novex tris-glycine SDS-PAGE gel for 50 min at 200 V and stained using InstantBlue Coomassie Stain (Expedeon, Cambridge, U.K.). The gels were photographed and the density of FliC bands compared using the gel analyser function of ImageJ v1.52n. The 55 kDa band from the Color Prestained Protein Standard, Broad Range (NEB, Hitchin, U.K.) was used to normalise for staining differences between gels.
4
SDS-PAGE Protein Purity Analysis
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SDS-PAGE was performed using 16% Tris-glycine gels in an XCell SureLock system (Thermo Fisher Scientific). Samples were loaded with final concentration of 1x SDS-PAGE loading buffer. For reduced samples, 100 mM 2-mercaptoethanol was added. SDS-PAGE gels were run at 190 V in 25 mM Tris-HCl, 192 mM glycine, 0.1% (w/v) SDS, pH 8.5. Gels were stained with InstantBlue Coomassie stain (Expedeon), destained with MilliQ water, and imaged using ChemiDoc XRS imager and analyzed with ImageLab (version 6.0.1) (Bio-Rad). In ImageLab, low sensitivity band detection in the final eluted lane (T) was calculated and compared with the protein control lane (Protein) at background subtraction of disk size 2 mm. Percentage purity is defined as 100 × [target protein Band % in lane T/target protein Band % in lane Protein].
5
SDS-PAGE Analysis of SpyCatcher-mi3 Conjugation
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SDS-PAGE was performed
with 12 or 18% Tris-glycine gels using an XCell SureLock system (Thermo
Fisher Scientific). Protein samples were loaded with 6× SDS-PAGE
loading buffer [0.23 M Tris·HCl pH 6.8, 24% (v/v) glycerol, 120
μM bromophenol blue, 0.23 M SDS] and heated for 3 min at 99
°C before loading on the gel. On reduced samples, loading buffer
contained 170 μM 2-mercaptoethanol. SDS-PAGE was performed at
200 V in 25 mM Tris·HCl and 192 mM glycine, 0.1% (w/v) SDS, pH
8.5. Gels were stained with InstantBlue Coomassie stain (Expedeon),
destained using Milli-Q water, and imaged using ChemiDoc XRS imager
and ImageLab (version 5.2) software (BioRad). ImageLab was also used
for band quantification. To assess the percentage of SpyCatcher-mi3
particle that had reacted with antigen, a sample of unreacted SpyCatcher-mi3
at the same starting concentration was run on the gel and defined
as 100% unreacted. % Conjugation was defined as 100 × [1 –
(SpyCatcher-mi3 band after antigen incubation)/ (SpyCatcher-mi3 band
in the absence of antigen)].
with 12 or 18% Tris-glycine gels using an XCell SureLock system (Thermo
Fisher Scientific). Protein samples were loaded with 6× SDS-PAGE
loading buffer [0.23 M Tris·HCl pH 6.8, 24% (v/v) glycerol, 120
μM bromophenol blue, 0.23 M SDS] and heated for 3 min at 99
°C before loading on the gel. On reduced samples, loading buffer
contained 170 μM 2-mercaptoethanol. SDS-PAGE was performed at
200 V in 25 mM Tris·HCl and 192 mM glycine, 0.1% (w/v) SDS, pH
8.5. Gels were stained with InstantBlue Coomassie stain (Expedeon),
destained using Milli-Q water, and imaged using ChemiDoc XRS imager
and ImageLab (version 5.2) software (BioRad). ImageLab was also used
for band quantification. To assess the percentage of SpyCatcher-mi3
particle that had reacted with antigen, a sample of unreacted SpyCatcher-mi3
at the same starting concentration was run on the gel and defined
as 100% unreacted. % Conjugation was defined as 100 × [1 –
(SpyCatcher-mi3 band after antigen incubation)/ (SpyCatcher-mi3 band
in the absence of antigen)].
6
Protein Separation and Detection Protocols
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HeLa cell lysates (15 μg), raw and solubilised E. coli membranes, affinity and size exclusion chromatography fractions, or purified pore proteins (2.2 ng) were separated on NuPAGE Novex 4–12% BisTris SDS gels (Life Technologies) and where indicated, stained with Instant Blue coomassie stain (Expedeon).
For Western blotting, proteins were transferred to PVDF (BioRad or iBlot, Life Technologies) membranes according to standard procedures. The primary antibodies used were anti-GFP (rabbit polyclonal α-GFP, A11122 Life Technologies, 1 in 1000) and anti-TPC2 (rabbit polyclonal α-TPC2, Eurogentec custom antibody, 1 in 1000)24 (link). Blots were developed using a secondary antibody (goat α-rabbit IgG/horseradish peroxidase (HRP) conjugate, 1706515 BioRad, 1 in 2000) and the ECL Prime Western Blotting System (GE Healthcare). All antibodies were incubated for 1 hr at room temperature. His-tagged proteins were detected using a monoclonal α-poly-histidine/alkaline phosphatase conjugate antibody (mouse, A5588 Sigma, 1 in 2000, 2 hrs at room temperature). Blots were developed using SIGMAFAST BCIP/NBT tablets (Sigma), as per the manufacturer’s instructions.
For Western blotting, proteins were transferred to PVDF (BioRad or iBlot, Life Technologies) membranes according to standard procedures. The primary antibodies used were anti-GFP (rabbit polyclonal α-GFP, A11122 Life Technologies, 1 in 1000) and anti-TPC2 (rabbit polyclonal α-TPC2, Eurogentec custom antibody, 1 in 1000)24 (link). Blots were developed using a secondary antibody (goat α-rabbit IgG/horseradish peroxidase (HRP) conjugate, 1706515 BioRad, 1 in 2000) and the ECL Prime Western Blotting System (GE Healthcare). All antibodies were incubated for 1 hr at room temperature. His-tagged proteins were detected using a monoclonal α-poly-histidine/alkaline phosphatase conjugate antibody (mouse, A5588 Sigma, 1 in 2000, 2 hrs at room temperature). Blots were developed using SIGMAFAST BCIP/NBT tablets (Sigma), as per the manufacturer’s instructions.
7
Profiling Antibody Glycosylation via Mass Spectrometry
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1 µg antibodies were digested with PNGaseF following manufacturer’s instructions (NEB, cat. #P0704). Samples were reduced with 5% β-mercaptoethanol before performing SDS-PAGE. Proteins were identified using InstantBlue™ Coomassie stain (Expedeon, cat. #ab119211). PNGaseF assays were performed in triplicate.
For mass spectrometry, pN6 antibody was trypsin-digested and analyzed by liquid chromatography–electrospray ionization–mass spectrometry as described in Teh et al. [33 (link)]. Briefly, samples were resuspended in 80 mM ammonium formiate buffer and run on a BioBasic C18 column with a 5% to 40% 80%-acetonitrile for 45 min, followed by a 15 min gradient from 40 to 90% 80%-acetonitrile, that facilitates elution of large peptides, at a flow rate of 6 µL/min. Peptide identification was performed with maXis 4G ETD (Bruker, Germany) in positive ion mode. Manual glycopeptide searches were made using DataAnalysis 4.0 (Bruker).
For mass spectrometry, pN6 antibody was trypsin-digested and analyzed by liquid chromatography–electrospray ionization–mass spectrometry as described in Teh et al. [33 (link)]. Briefly, samples were resuspended in 80 mM ammonium formiate buffer and run on a BioBasic C18 column with a 5% to 40% 80%-acetonitrile for 45 min, followed by a 15 min gradient from 40 to 90% 80%-acetonitrile, that facilitates elution of large peptides, at a flow rate of 6 µL/min. Peptide identification was performed with maXis 4G ETD (Bruker, Germany) in positive ion mode. Manual glycopeptide searches were made using DataAnalysis 4.0 (Bruker).
8
SDS-PAGE Analysis of Radiolabeled Proteins
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CFPS reactions were supplemented with 10 µM of radioactive 14C-Leu. Three microlitre of the soluble fraction of each reaction was loaded on a 4–12% NuPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer (Invitrogen, Carlsbad, CA, USA) after heat-denaturation and reduction by 1,4-dithiothreitol (DTT). The gel was stained using InstantBlue Coomassie stain (Expedeon, San Diego, CA, USA) for 1 h, destained overnight, soaked in Gel Drying Solution (Bio-Rad, Hercules, CA, USA) for 30 min, fixed with cellophane films, dried overnight in GelAir Dryer (Bio-Rad, Hercules, CA, USA) and exposed for 72 h on Storage Phosphor Screen (GE Healthcare Biosciences, Pittsburgh, PA, USA). The autoradiogram was scanned using a Typhoon FLA7000 Imager (GE Healthcare Biosciences, Pittsburgh, PA, USA).
9
Ubiquitin Topoisomer Preparation and Analysis
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Ubiquitin monomer, BSA, Tris and DTT were purchased from Sigma Aldrich. Di-Ubiquitin topoisomers (linear, K6, K11, K27, K29, K33, K48 and K63-linked ubiquitin-ubiquitin) were purchased from Boston Biochem. Complete protease inhibitors cocktail tablets were obtained from Roche. Protein G–Sepharose, glutathione–Sepharose and ECL reagents were from GE Healthcare. Doxycycline, DMSO, BSA, 3-amino, 1,2,4 triazole and benzamidine were from Sigma–Aldrich. PMSF was from Melford. Novex 4–12% polyacrylamide Bis-Tris gels, LDS sample buffer, puromycin, hygromycin, blasticidin and other tissue culture reagents were from Invitrogen Life Technologies. Instant Blue Coomassie stain was from Expedeon. PEI (polyethylenimine) was from Polysciences.
10
SDS-PAGE Protein Analysis
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Samples were mixed with 5× SDS-PAGE loading buffer [0.23 M Tris-HCl pH 6.8, 24% (v/v) glycerol, 120 µM bromophenol blue, 0.23 M sodium dodecyl sulfate, 100 mM 2-mercaptoethanol], heated for 3 min at 99 °C, and loaded onto a 16% Tris-glycine gel. Gels were run in an XCell SureLock system (Thermo Fisher) for 60 min at 190 V, stained with InstantBlue Coomassie stain (Expedeon), destained with MilliQ water, and imaged using a ChemiDoc XRS imager. Gel densitometry was performed with ImageLab 6.0.1 (Bio-Rad).
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