A Western blot was used to verify the deletion of toxA in PGN5 by the absence of the Exotoxin A product. Cells from 24‐h broth cultures of P. aeruginosa strains PAO1, Exotoxin A‐positive PA103 and PGN5 were removed by centrifugation. Supernatant was filter‐sterilized with a 0.2 μm syringe filter and concentrated with an Amicon Ultra‐15 Centrifugal Filter Unit with a 10‐kDa cut‐off (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Total protein concentration of the supernatant (extracellular fraction) was quantified with the bicinchoninic acid assay (BCA) using a bovine serum albumin (BSA) standard (Pierce, Rockford, IL, USA). For Coomassie staining, equal amounts of protein (60 mg) were run with SDS‐PAGE on a TGX 4‐20% gel (Bio‐Rad, Hercules, CA, USA), stained with Coomassie R‐250 (Protea Biosciences, Morgantown, WV, USA) and imaged on a ProteinSimple Fluorochem M (San Jose, CA, USA). For Exotoxin A western blot, samples with equal total protein concentrations (0.05 μg/μL) were run on the ProteinSimple Wes automated Western blot system using the 12–230 kDa separation module and the 8 × 25 capillary cartridge (San Jose, CA, USA). Exotoxin A was detected with a polyclonal rabbit anti‐Exotoxin A antibody (Sigma, St. Louis, MO, USA) diluted 1:200 and the ProteinSimple anti‐rabbit detection module (San Jose, CA, USA).
Tgx 4 20 gel
Manufactured by Bio-Rad
Sourced in United States
TGX 4–20% gels are pre-cast polyacrylamide gels designed for protein separation and analysis. They feature a gradient of 4% to 20% polyacrylamide concentration, which allows for the separation of a wide range of protein molecular weights. These gels are compatible with standard SDS-PAGE techniques.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Epr16897 ab179483, by Abcam (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay (ripa), by Boston BioProducts (2 mentions)
Western lightning, by PerkinElmer (2 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Coomassie brilliant blue staining, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Westernbright quantum hrp substrate, by Advansta (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Monoclonal anti c myc antibody, by BioLegend (1 mentions)
12 protocols using tgx 4 20 gel
1
Western Blot Validation of toxA Deletion
2
SDS-PAGE Analysis of CD160 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD160-containing supernatant from HEK293T cells and mouse IgG2a Fc purified protein were run by SDS-PAGE alongside a purified commercial source of CD160-His (Sino Biological). 1 µg of purified protein or 5 µl of supernatant were run on to a TGX 4–20% gel according to the manufacturer’s protocols (Bio-Rad). Samples were prepared as follows: 5 µL of sample loading buffer, 2 µL of reducing buffer (not included under non-reducing conditions), made up to 20 µL final volume with PBS. Following SDS-PAGE, the gel was covered with Coomassie R-250 stain solution (Sigma-Aldrich) and stained for 60 min with gentle agitation on a rotating plate shaker. Staining solution was decanted and the gel was destained (dH2O, methanol, and acetic acid in a ratio of 50/40/10 (v/v/v)) until the protein bands became visible. Images of the proteins gel were acquired using QuantStudio imager (GE Lifesciences).
3
Protein Extraction and Immunoblotting Protocol
4
Sortase-Mediated Protein Conjugation of ELP-CCMV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Standard ELP-CCMV and VY1-VY8 ELP-CCMV capsids were dialyzed at 4 °C towards PBS (500 mM NaCl) and pH 7.5 buffer (100 mM NaCl) respectively to ensure disassembly. ELP-CCMV variants and GFP were then buffer exchanged towards sortase buffer by spin-filtration (Amicon® Ultra 0.5 mL). Dimers (Standard ELP-CCMV) or capsids (VY1-VY8 ELP-CCMV), sortase A and GFP were incubated in equimolar concentrations (30 μM) at 21 °C, 400 rpm up to 24 h. Reaction mixtures at indicated time points were analyzed by SDS-PAGE using Mini-Protean TGX 4–20% gels (tris/glycine buffer) for 1 hour at 150 V. Proteins were visualized via Coomassie Brilliant Blue staining (Bio-Rad). ImageJ analysis software was used to calculate the conversion percentage using the following formula, where gel is the intensity of the protein band on the SDS-PAGE gel and MW is the molecular weight of the protein of interest:![]()
5
Quantifying Hippocampal HIF-1α Expression
6
Immunoblotting of Skeletal Muscle Proteins
7
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were performed using an adapted chloroacetic acid (TCA) method. Briefly, cells from 5- to 10 ml aliquots of cultures were harvested, and 1 ml of 20% TCA was added. The supernatant was removed after centrifugation, and the pellet was resuspended in 100 μl of 20% TCA and stored at −80°C for 1 hr. Samples were thawed on ice, glass beads were added, and cells were broken using a FastPrep FP120 cell disrupter (BIO 101 ThermoSavant, Obiogene, Carlsbad, CA). The lysate was recovered by punching a hole on the bottom of the tube, and the glass beads were further washed with 200 μl of 5% TCA. Lysates were centrifuged at 1000 × g for 3 min, and the pellet was thoroughly resuspended in 100 μl of 2 × Laemmli buffer and 50 μl of 2 M Tris base. After boiling for 5 min, 10–20 μl were loaded in the gels.
For general purposes, TGX (4–20%) gels from BioRad were used, at constant 100 v running conditions. To detect the phosphorylated forms of Kap123, 8% acrylamide/0.1% bisacrylamide, pH 9.2 custom-made gels were used at constant 5mA running conditions.
Western blots were repeated from at least three independent experiments in each case.
For general purposes, TGX (4–20%) gels from BioRad were used, at constant 100 v running conditions. To detect the phosphorylated forms of Kap123, 8% acrylamide/0.1% bisacrylamide, pH 9.2 custom-made gels were used at constant 5mA running conditions.
Western blots were repeated from at least three independent experiments in each case.
8
Quantifying Hippocampal HIF-1α Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis for HIF-1α was performed using methods described previously (38 (link)). Briefly, protease inhibitors were added to hippocampal homogenates (n = 4/group) used for ascorbate and GSH assays. Protein concentration was determined by bicinchoninic acid assay and 35 μg protein aliquots were loaded onto pre-cast TGX 4–20% gels (BioRad, Hercules, CA), separated electrophoretically, then transferred onto polyvinylidene difluoride membranes (Millipore Immobilon, Bedford, MA). Membranes were incubated with a rabbit monoclonal antibody against HIF-1α (1:800 dilution; [EPR16897] ab179483); Abcam, Cambridge, MA) overnight at 4°C then appropriate secondary antibody for 2 hours at room temperature. Protein bands were visualized with Supersignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL) and imaged on a BioRad Chemi Doc XRS+ imaging system with band intensity analyzed by ImageLab Software (Version 4.0, BioRad). Membranes were stripped and re-probed with antibody against β-actin (1:2,000) using the methods above. HIF-1α densitometry measurements were normalized to corresponding actin densitometry measurements within each lane. Fold-increase in HIF-1α was determined using corresponding group-specific shams from the same membrane.
9
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were prepared in radioimmunoprecipitation assay buffer (RIPA, Boston BioProducts), separated by SDS-PAGE at 100V using a Bio-Rad 4–20% TGX gel (Bio-Rad), blocked with 5% Milk-TBS-T for standard western blotting, 5% BSA for phospo-VEGFR2 blotting, and probed with antibodies overnight at 4°C. Peroxidase-conjugated secondary antibodies were incubated at RT for 1 hour, and bands were exposed with Western Lightning (PerkinElmer). Blots were imaged on a Fluorchem M (ProteinSimple). All antibodies and dilutions are presented in Supplementary Table 3 . Uncropped western blots are presented in Supplementary Fig. 11 .
10
Western Blot Analysis of Cell Lysates
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!