Lysates of nonadherent EAEC were prepared by suspending bacterial pellets in reducing SDS‐PAGE sample buffer. For adherent EAEC, infected T84 monolayers were lysed in ice‐cold lysis buffer (50 mM Hepes pH 7.4, 50 mM NaCl, 1% Triton X‐100) containing protease inhibitor cocktail (1:200, Sigma). After heat denaturation, proteins were separated in 15% SDS‐polyacrylamide gels and transferred to PVDF membranes (VWR) using a Mini‐PROTEAN Tetra Cell device (Bio‐Rad). Membranes were blocked in 5% skimmed milk in TBS/0.05% Tween‐20 for 1 hr and incubated in polyclonal rabbit anti‐dispersin (1:5,000; kindly provided by Christopher Icke, University of Birmingham) overnight at 4°C followed by HRP‐conjugated goat anti‐rabbit IgG (1:20,000; Sigma) for 30 min. Membranes were developed by enhanced chemiluminescence (Immobilon Western, Millipore) and imaged with a FluorChem E Imager (ProteinSimple). Densitometric analysis of band intensities was performed using ImageJ software (https://imagej.nih.gov/ij /).
Mini protean tetra cell device
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN Tetra Cell is a vertical electrophoresis system designed for polyacrylamide gel electrophoresis (PAGE) analysis. It has a compact design and can run up to four gels simultaneously. The device is compatible with a range of gel sizes and can be used for a variety of applications, including protein separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Pvdf membrane, by Avantor (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Gs 800 calibrated imaging densitometer, by Bio-Rad (1 mentions)
Thermomixer comfort device, by Eppendorf (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using mini protean tetra cell device
1
Dispersin Detection in EAEC Lysates
2
SDS-PAGE Protein Separation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were mixed with 4X electrophoresis sample buffer (0.125 M Tris–HCl pH 6.8, 4% SDS; 20% v/v glycerol, 0.2 M DTT, 0.01% bromophenol blue). SDS-PAGE was performed in 10% polyacrylamide gels using a Mini-PROTEAN Tetra Cell device (BioRad, Hercules, CA, USA) according to the Laemmli method [32 (link)] at 120 V for stacking gel and 180 V for the resolving gel. The electrophoresis gels were visualized with the use of a staining solution containing Coomassie brilliant blue (CBB).
3
SDS-PAGE Analysis of GCF Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GCF protein extracts were subjected to SDS-PAGE analysis under reducing conditions. An aliquot of each pool (8 µg of protein content) was combined with 2× Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue) including 0.5% 2-mercaptoethanol. Protein denaturation was obtained by heating the mixture at +95 °C for 5 min in a Thermomixer Comfort device (Eppendorf, Milan, Italy). Protein separation was then carried out using precast gel BoltTM 12% Bis-Tris Plus and 4–12% Bis-Tris Plus, 12 wells. The electrophoretic run was performed in a Mini Protean Tetra Cell device (Bio-Rad, Hercules, CA, USA) using MES SDS 1× as the running buffer, at 100 V for 30 min, and next at 200 V for the remaining run time. After rinsing with pure water, the gels were stained overnight at constant moderate shaking with Coomassie Blue G-250, and de-stained with 30% methanol/10% glacial acetic acid solution. The GS-800 Calibrated Imaging Densitometer (Bio-Rad, Hercules, CA, USA) was used to acquire the gel images.
4
Supinoxin Modulates Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 and H69AR cells were seeded and allowed to adhere for 24 hours in 6-well plates. Supinoxin was dissolved in DMSO in order to prepare a stock solution at 100μM. Cells were treated with different concentrations of Supinoxin (0, 20, 70, or 100 nM) diluted in appropriate growth media for 24 to 48 hours. Cells were then trypsinized and collected using 1X TrypLE Express (Catalog No: 12604013) followed by lysis in Cell Lysis Buffer (Cell Signaling, 9803), with EDTA-free protease inhibitor cocktail (Roche, 11873580001). 20–50 mg of total protein was loaded onto an 8% SDS-PAGE gel, which was then electrophoresed and transferred to nitrocellulose membrane using the Mini PROTEAN Tetra Cell device (Bio-Rad). After being blocked in 2% skimmed milk for 5 minutes, membranes were incubated at 4°C overnight with primary antibodies β-catenin (Thermo (Invitrogen) CAT-5H10), DDX5 (Abcam, ab126730), RFC1 (Abcam, ab3853), phosphor-DDX5 (Y593) (1:500 Abcam, ab62255), c-Myc (Cell Signaling D84C12), β-Actin (Sigma A5441). HRP-conjugated secondary antibodies were used to image western blots: anti-rabbit (Thermo 31460) anti-mouse (Jackson 115-035-003). The blots were detected using Milipore Immobilon Crescendo Western HRP Substrate reagent (Catalog No: WBLUR0500).
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!