The protocol for conjugating dUb and hE2 via an isopeptide bond was adapted from (Plechanovová et al. 2012 (link)). Briefly, a 30 mL reaction mixture containing 50 mM Tris, pH 10, 150 mM NaCl, 5 mM MgCl2, 0.8 mM TECP, 200 µM dUb-His6, 1 µM human E1-His6 (Berndsen and Wolberger 2011 (link)), 180 µM hE2, 3 mM ATP was incubated for 24 h at 35 °C. To remove unconjugated hE2, the reaction mixture was added to 3 mL of HisLink™ resin (Promega) equilibrated with buffer A with 20 mM Imidazole and incubated for 30 min at 4 °C. Conjugated hE2–dUb-His6 was then eluted from the beads using buffer A with the addition of 150 mM imidazole (pH 8.0) and dialysed overnight in buffer A at 20 °C with Thrombin (Sigma). The post-Thrombin cleavage sample was passed through a HisLink™ resin to remove any remaining His6-tag material, while the flow-through fractions were injected to a HighLoad 16/600 Superdex 75 (Cytiva) column equilibrated with 50 mM Tris, 150 mM NaCl, 0.5 mM TCEP to isolate hE2–dUb. The final purity of the conjugated proteins was assessed by SDS-PAGE stained with InstantBlue (Sigma) and concentrated using Amicon centrifugal filter unit (Millipore). The sample concentration was quantified by UV absorption at 280 nm and calculated using an extinction coefficient of 1.043.
Instantblue
Manufactured by Merck Group
Sourced in United States, France
InstantBlue is a fast, convenient, and easy-to-use protein staining solution. It is designed for the rapid visualization and quantification of proteins in polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (3 mentions)
Mouse poly his antibody, by Qiagen (3 mentions)
Tgx precast gel, by Bio-Rad (3 mentions)
Typhoon 5 imaging system, by Cytiva (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Iblot system, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (2 mentions)
Ni nta agarose bead, by Qiagen (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gs c 18 antibody, by Bio-Rad (2 mentions)
Amicon centrifugal filter unit, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Hislink resin, by Promega (1 mentions)
Thrombin, by Cytiva (1 mentions)
Kta purifier, by GE Healthcare (1 mentions)
Hitrap sp ff column, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
43 protocols using instantblue
1
Enzymatic Conjugation of Ubiquitin and E2
2
Flagella SDS-PAGE Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After PEG precipitation, flagella were boiled in LDS and run on a 4%–12% Bis-Tris gel (Thermo-Fisher, Hillsboro, OR, United States of America) and stained with InstantBlue (Sigma-Aldrich, Taufkirchen, Germany).
3
Thermal Stability of P-gp Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Membrane vesicles containing WT or 14A mutant P-gp were incubated at a range of temperatures (37–80 °C) for 10 min in the presence or absence of ATP as described previously (23 (link)). Next, 5× gel sample buffer was added and incubated for 20 min at 37 °C. Samples were then run on a 7% Tris-Acetate NuPAGE gel and stained with InstantBlue (Sigma-Aldrich).
4
Gel Fractionation for Protein Separation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gel fractionation was performed by putting 70 µL of reaction mix per well into a 10-well Bolt NuPAGE 4–12% Bis-Tris gel (Invitrogen, Cat #: NW04120BOX) and running at 200 Volts for 22 min in MES Buffer. It was soaked in Instant Blue (Sigma, Darmstadt, Germany, Cat #: 1SB1L) dye for 15 min to stain the gel. The gel was divided horizontally into ten equal-sized strips (Figure S1 ) and then vertically, with the lanes of each protein sample serving as a guide. Using a clean scalpel, the gel slices were then transferred to the wells of a 96-well plate. To sufficiently destain, the pieces were repeatedly washed with 50 mM ammonium bicarbonate and 50 mM ammonium bicarbonate/50% acetonitrile. Each washing cycle took 10 min on a shaking rotator. After the final wash, the gel pieces were dried for 10 min at 50 °C in a centrifugal evaporator.
5
Purification of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were expressed according to a previously published protocol (41 (link)). Bacteria were lysed using a Stansted Fluid Power pressure cell homogenizer (100 MPa). Proteins harboring His tags were purified by nickel affinity chromatography as described elsewhere (29 (link)). Proteins without His tags were purified by cation exchange chromatography (CIEX) using a 5-ml HiTrap SP-FF column on a fast protein liquid chromatography (FPLC) device (Äkta purifier; GE Healthcare Life Sciences). Proteins used for the in vivo experiments were expressed in E. coli ClearColi BL21(DE3) as described elsewhere (41 (link)). In this case, protein purification was performed in an endotoxin-free environment. The purification columns and chromatography system were decontaminated using 1 M NaOH prior to sample loading. Additional purification by size exclusion chromatography (SEC) was performed following the CIEX step (Superdex 200 Increase 10/300 GL; GE Healthcare) using SEC running buffer (50 mM Na2HPO4, 500 mM NaCl, pH 7.4). Proteins were tested for endotoxin content using an EndoZyme kit (Hyglos, Regensburg, Germany) according to the manufacturer’s instructions. Protein identity and purity were confirmed by SDS-PAGE, followed by Coomassie staining (InstantBlue; Sigma).
6
Proteasomal Degradation of α-Synuclein and Enolase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human α-synuclein
(rPeptide) or yeast enolase (Sigma-Aldrich) was incubated with human
20S proteasome activated with 0.01% SDS. The h20S/protein substrate
ratio was 1:100 pmol and 1:10 pmol for α-synuclein and enolase,
respectively. Degradation experiments were carried out at 37 °C
in 20 mM HEPES, pH 7.4, at a 10 μL total sample volume for 1
h (α-synuclein) or 4 h (enolase). Either DMSO (control) or compounds
dissolved in DMSO were added to evaluate the influence of PR peptides
on the degradation process. The DMSO concentration never exceeded
0.05%. The reaction was stopped with 4× Laemmli buffer, and then
samples were boiled for 5 min at 95 °C and loaded (8 μL)
onto a 12% (α-synuclein) or 10% (enolase) SDS–PAGE gel.
The protein bands were detected with Coomassie Blue-based reagent
(InstantBlue, Sigma-Aldrich). Quantitative image analysis was carried
out with Quantity One 1-D analysis software (Bio-Rad). The amount
of the nondegraded protein was calculated after reduction of the background
intensity and expressed as a percentage of the control. Each value
represents an average of at least three experiments. All results are
presented as a mean ± SEM. Statistical analysis was performed
using SigmaPlot 12.3 and one-way ANOVA followed by a Bonferroni post
hoc test for pairwise comparison. A P value of <0.05
was considered statistically significant.
(rPeptide) or yeast enolase (Sigma-Aldrich) was incubated with human
20S proteasome activated with 0.01% SDS. The h20S/protein substrate
ratio was 1:100 pmol and 1:10 pmol for α-synuclein and enolase,
respectively. Degradation experiments were carried out at 37 °C
in 20 mM HEPES, pH 7.4, at a 10 μL total sample volume for 1
h (α-synuclein) or 4 h (enolase). Either DMSO (control) or compounds
dissolved in DMSO were added to evaluate the influence of PR peptides
on the degradation process. The DMSO concentration never exceeded
0.05%. The reaction was stopped with 4× Laemmli buffer, and then
samples were boiled for 5 min at 95 °C and loaded (8 μL)
onto a 12% (α-synuclein) or 10% (enolase) SDS–PAGE gel.
The protein bands were detected with Coomassie Blue-based reagent
(InstantBlue, Sigma-Aldrich). Quantitative image analysis was carried
out with Quantity One 1-D analysis software (Bio-Rad). The amount
of the nondegraded protein was calculated after reduction of the background
intensity and expressed as a percentage of the control. Each value
represents an average of at least three experiments. All results are
presented as a mean ± SEM. Statistical analysis was performed
using SigmaPlot 12.3 and one-way ANOVA followed by a Bonferroni post
hoc test for pairwise comparison. A P value of <0.05
was considered statistically significant.
7
Instant Protein Gel Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE gels were placed into 10mL of InstantBlue™ (Sigma ISB1L) for 1–16 h with gentle rocking. Gels were then rinsed for 4 × 15 min before imaging on the LI-COR Odyssey system. Gels used for mass spectrometry were stained as detailed in the sample preparation and mass spectrometry methods section.
8
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were loaded on 4% to 12% Bis-Tris SDS-polyacrylamide gels and stained with Instant Blue (Sigma-Aldrich, St. Louis, MO). For immunoblotting, the gels were transferred to nitrocellulose membranes using the iBlot system (ThermoFisher, Waltham, MA). Immunoblotting was performed using the iBind system (ThermoFisher). 1D4 antibody in 4% paraformaldehyde in PBS (1:10,000) was used for the primary antibody, and IRDye800TM-conjugated affinity purified goat anti-mouse immunoglobulin G (Licor, Lincoln, NE), diluted 1:10,000, was used for the secondary antibody. The membrane was then washed with iBind Solution and scanned using an Odyssey Infrared imager (Licor, Lincoln, NE).34 (link),36 (link) Fluorescence intensity was measured using Photoshop CC. A one-way analysis of variance (ANOVA) with Tukey's multiple comparison test was used to establish statistical significance, with P < 0.05 considered significant.
9
Native Polyacrylamide Gel Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Native polyacrylamide gels were prepared by layering 12.5%, 7.5% and 5% concentration separating polyacrylamide gel layers and topping them with 3% stacking polyacrylamide gel without SDS. The SDS was also omitted from standard Tris-glycine running buffer. Aliquots of 2.5 μl of in vitro translation reactions were mixed with native loading buffer (60 mM Tris–HCl pH 6.8, 10% glycerol) deposited in pre-washed wells and migrated in pre-chilled buffer in the cold room overnight at constant 30 V current. Gel was stained using InstantBlue™ (Sigma-Aldrich), or transferred onto nitrocellulose membrane for detection with streptavidin-alkaline phosphatase conjugate (catalog #S921, Invitrogen). Bands at the size corresponding to biotin signal were excised from stained gel using sterile scalpel, digested with Trypsin/LysC and analysed by mass spectrometry.
10
SDS-PAGE and Western Blot Analysis of ZIKV VLPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
sE samples were subjected to 10% SDS-PAGE, and the fractionated proteins were detected by direct staining of the gel with InstantBlue (Sigma) or by Western blotting. VLP samples were separated by 10% or 14% SDS-PAGE, blotted to a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL; Merck Millipore), blocked overnight with ODYSSEY blocking buffer (LI-COR), and then incubated with DIII1B antibody (anti-ZIKV E DIII generated in-house as described in Fig. 3 ) or ZIKA prM antibody (GeneTex) for 1 h. This was followed by incubations with anti-mouse IgG (IRDye 800CW; LI-COR) and anti-rabbit IgG (IRDye 680RD; LI-COR). Images were acquired by a LICOR machine.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!