Antiserum to whole-cell B. uniformis 8492 was prepared in rabbits by Lampire BioLogical Laboratories using the Express-Line polyclonal antiserum protocol. To create an antibody fraction specific to the molecule lost in B. uniformis 8492 tn::00969, we performed an antibody adsorption to remove antibodies reactive to the transposon mutant as previously described (46 (link)). For PAGE analysis, bacteria or purified LPS was boiled in lithium dodecyl sulfate (LDS) sample buffer and subjected to electrophoresis using NuPAGE 12% bis-Tris polyacrylamide gels with MES (morpholineethanesulfonic acid) buffer (Life Technologies). In some cases, the gel was subjected to silver staining (Pierce silver stain kit; Thermo Scientific) according to manufacturer protocols. For Western immunoblot analyses, the contents of the gels were transferred to polyvinylidene difluoride (PVDF) membranes and probed with the adsorbed serum. Alkaline phosphatase-labeled anti-rabbit IgG (Pierce) was the secondary antibody, and the membranes were developed with BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate/Nitro Blue Tetrazolium) (KPL, Gaithersburg, MD).
Nupage 12 bis tris polyacrylamide gel
Manufactured by Thermo Fisher Scientific
The NuPAGE® 12% Bis-Tris polyacrylamide gel is a pre-cast protein electrophoresis gel used for the separation and analysis of proteins. It is a 12% Bis-Tris formulation designed for the resolution of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Xcell 2 blot module for wet tank transfer, by Thermo Fisher Scientific (2 mentions)
Tmbz 3 3 5 5 tetramethylbenzidine, by Merck Group (2 mentions)
0 25 m sodium acetate ph 5, by Merck Group (2 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg antibody, by Merck Group (1 mentions)
Nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
Protein sample buffer, by Merck Group (1 mentions)
Facscanto analyzer, by BD (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Ezblue coomassie brilliant blue g 250, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
7 protocols using nupage 12 bis tris polyacrylamide gel
1
Antibody Adsorption and Analysis of B. uniformis LPS
2
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SDS-PAGE was performed using sodium dodecyl sulfate (SDS) NuPAGE® 12% Bis–Tris polyacrylamide gels (Life Technologies™) with NuPAGE® morpholinepropanesulfonic acid (MOPS) buffer at 180 V for 60 min. Before loading the gel with 15 μL of culture supernatant, each sample supernatant was correlated to the wet cell weight of each sample using RO water.
After SDS-PAGE, the separated proteins were transferred to a nitrocellulose membrane using the XCell II™ Blot Module for wet (tank) transfer (Life technologies™) according to the manufacturer’s instructions. The HyHEL-Fab was immunologically detected with Anti-Human IgG antibody (Abcam, ab7497) produced in mouse, specifically recognizing the Hinge region of Human IgG and Anti-Mouse IgG antibody (Sigma-Aldrich, A3673) coupled with HRP, which was produced in goat. Carboxylesterase was immunologically detected with rabbit anti-CES antiserum (produced in rabbit, provided by Biomin Holding GmbH) and carboxypeptidase Y was detected using anti-CPY antiserum (produced in rabbit, kindly provided by Günther Daum/Karlheinz Grillitsch, Graz University of Technology). As secondary antibodies, anti-rabbit IgG antibodies coupled with horseradish peroxidase (produced in goat, Sigma-Aldrich, A0545) were used.
After SDS-PAGE, the separated proteins were transferred to a nitrocellulose membrane using the XCell II™ Blot Module for wet (tank) transfer (Life technologies™) according to the manufacturer’s instructions. The HyHEL-Fab was immunologically detected with Anti-Human IgG antibody (Abcam, ab7497) produced in mouse, specifically recognizing the Hinge region of Human IgG and Anti-Mouse IgG antibody (Sigma-Aldrich, A3673) coupled with HRP, which was produced in goat. Carboxylesterase was immunologically detected with rabbit anti-CES antiserum (produced in rabbit, provided by Biomin Holding GmbH) and carboxypeptidase Y was detected using anti-CPY antiserum (produced in rabbit, kindly provided by Günther Daum/Karlheinz Grillitsch, Graz University of Technology). As secondary antibodies, anti-rabbit IgG antibodies coupled with horseradish peroxidase (produced in goat, Sigma-Aldrich, A0545) were used.
3
Carboxylesterase Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10 μL of culture supernatant (containing secreted carboxylesterase enzyme) were run on a reducing sodium dodecyl sulfate (SDS) NuPAGE® 12% Bis-Tris polyacrylamide gel (Life technologies™) with NuPAGE® morpholinepropanesulfonic acid (MOPS) buffer at 180 V for 60 min. Protein bands were visualized using Coomassie staining solution. For Western blotting, SDS-PAGE separated proteins were transferred to a nitrocellulose membrane using the XCell II™ Blot Module for wet (tank) transfer (Life technologies™) according to the manufacturer’s instructions. Carboxylesterase was detected using anti-carboxylesterase antiserum as described by Heinl et al. [48 (link)].
4
Western Blotting and Flow Cytometric Analysis of Zta
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and boiled in a protein sample buffer (Sigma). The protein was resolved on a NuPAGE 12 % Bis-Tris polyacrylamide gel (Life Technologies) in 1× morpholinepropanesulfonic acid buffer (Invitrogen) at 200 V for 50 min. The proteins were transferred onto nitrocellulose membranes (Santa Cruz Biotechnology) and incubated overnight with the indicated antibodies at 4 °C. The following day, the membranes were incubated with IRDye secondary antibodies (LiCor), and the proteins were detected by fluorescence at 680 and 800 nm. In addition, the BZ1 mouse monoclonal antibody to Zta [47 (link)] and a rabbit polyclonal recognizing beta-Actin (Sigma) were used to detect proteins by Western blotting. Intracellular analysis of Zta expression was detected using BZ1 mouse monoclonal antibody to Zta [47 (link)] and a Fix and Perm Cell fixation and permeabilization system (Life Technologies) and subsequently detected using a FACS Canto analyzer (Beckton Dickinson).
5
Yeast Protein Profiling by SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used SDS-PAGE to allow direct visualisation of yeast protein profiles and detect a possible degradation during freezing treatment of yeast. Three treatments were compared -Lane A: protein extract from fresh live yeast, Lane B: protein extract from frozen yeast (stored at -20 °C for 175 days) and Lane C: protein extract digested with trypsin (V511A, Promega) to visualise a digested protein profile. Protein concentrations in extracts were adjusted to 5 μg of protein and were mixed with NuPAGE® LDS (4 x) sample buffer (Life Technologies) and DTT (50mM). The mix was heated on dry block at 80 °C for 10 min. Samples were then applied to precast NuPAGE® 12% Bis-Tris polyacrylamide gel (Life Technologies). NuPAGE® MES SDS ( 20x) was used as running buffer (Life Technologies) to resolve small molecular weight proteins. SDS-PAGE was then performed at 200 V for 40 min. After electrophoresis, gels were stained with EZBlue™ Coomassie Brilliant Blue G-250 (Sigma Aldrich). Unstained natural protein standard was used as a molecular weight marker.
6
Heme-based Protein Visualization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heme staining, based on heme peroxidase activity, was performed essentially as reported 56 (link). Protein samples were separated on a NuPAGE® Bis-Tris 12% polyacrylamide gel (Invitrogen). The gel was rinsed with water for 15 s and then incubated for 1 h in the dark in a solution containing 30 mL of 40 mM TMBZ (3,3,5,5′-Tetramethylbenzidine, Sigma-Aldrich) in methanol followed by the addition of 70 mL of 0.25 M sodium acetate pH 5.0 (Sigma-Aldrich). Then, 5 mL of 3% hydrogen peroxide were added and mixed well until a signal corresponding to the MBP Z-ISO band appeared. The gel background was removed by destaining 15 min with 3:7 isopropanol: 0.25 M sodium acetate.
7
Heme-based Protein Visualization Protocol
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!