Assay of PKM2 dimer and tetramer in vitro follows previous descriptions41 . In brief, after 40ng PKM2-FBP complex was treated with 200–300μM SNO-CoA for 10 min at room temperature, 5 μl fresh glutaraldehyde (50%) was added to a reaction mixture containing 100mM HEPES (pH 7.5) for 5 min at 37°C. The cross-linking reaction was terminated by addition of 5 μl 1M Tris-HCL (PH 8.0). Assay of PKM2 dimer and tetramer in situ was carried out as described previously14 (link). DSS (disuccinimidyl suberate; Thermo Scientific) (final 500 μM) was added to cells for 30 min at room temperature to cross-link proteins. Cells were lysed in RIPA Buffer and protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by 4–20% Criterion™ Precast Midi Protein Gel (BIO-RAD) and monomer, dimer and tetramer forms of PKM2 were detected with PKM2 antibody (sc-365684, Santa Cruz Biotechnology).
Criterion precast midi protein gel
Manufactured by Bio-Rad
Sourced in United States
The Criterion Precast Midi Protein Gel is a pre-cast polyacrylamide gel designed for protein separation and analysis. It provides a standardized and consistent format for electrophoresis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Hrp conjugated anti rabbit or anti mouse secondary antibody, by Vector Laboratories (3 mentions)
Pkm2 antibody, by Santa Cruz Biotechnology (2 mentions)
Dextran sulfate sodium (dss), by Thermo Fisher Scientific (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Protein assay dye reagent, by Bio-Rad (2 mentions)
Pierce supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (1 mentions)
7 protocols using criterion precast midi protein gel
1
In Vitro and In Situ Assay of PKM2 Oligomers
2
In Vitro and In Situ Assay of PKM2 Oligomers
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Assay of PKM2 dimer and tetramer in vitro follows previous descriptions41 . In brief, after 40ng PKM2-FBP complex was treated with 200–300μM SNO-CoA for 10 min at room temperature, 5 μl fresh glutaraldehyde (50%) was added to a reaction mixture containing 100mM HEPES (pH 7.5) for 5 min at 37°C. The cross-linking reaction was terminated by addition of 5 μl 1M Tris-HCL (PH 8.0). Assay of PKM2 dimer and tetramer in situ was carried out as described previously14 (link). DSS (disuccinimidyl suberate; Thermo Scientific) (final 500 μM) was added to cells for 30 min at room temperature to cross-link proteins. Cells were lysed in RIPA Buffer and protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by 4–20% Criterion™ Precast Midi Protein Gel (BIO-RAD) and monomer, dimer and tetramer forms of PKM2 were detected with PKM2 antibody (sc-365684, Santa Cruz Biotechnology).
3
Western Blot Quantification of Neuronal and Astrocytic Proteins
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Protein concentrations of cell lysate of neurons and astrocytes were determined by Protein Assay Dye Reagent (Bio-Rad). Equal amounts of proteins (10–20 μg) were loaded and separated on a 4%–15% Criterion Precast Midi Protein Gel (Bio-Rad) and then transferred to a 0.45 μm pore size polyvinylidene difluoride (PVDF) membrane and immunoblotted with the appropriate primary antibodies. Primary antibodies were incubated overnight, followed by washing and probing with the appropriate HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA). The immunoreactive bands were visualized by Pierce SuperSignal Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, IL) and captured by ChemiDoc Imaging System (BioRad, Hercules, CA). Saturated pixel highlighting feature of the instrument was used to prevent image overexposure for chemiluminescence. Band intensity was quantified using Image Lab 6.0.1 (Bio-Rad, Hercules, CA).
4
Western Blot Protein Analysis
5
Western Blot Quantification of Neuronal and Astrocytic Proteins
6
Western Blot Analysis of Tight Junction Proteins
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Protein concentrations of homogenized microvessel supernatants were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were loaded into a midi Criterion precast protein gel (4–20%, Bio-Rad, Hercules, CA, USA), separated by gel electrophoresis and subsequently transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) using the Trans-Blot Turbo system (Bio-Rad, Hercules, CA, USA). At room temperature, blots were blocked with 5% milk powder in PBS containing 0.1% Tween 20 for at least 1 h. Next, blots were incubated with primary antibodies for β-actin (1:2,000; #4970, Cell Signaling Technology, Danvers, MA, USA), claudin-5 (1:500; #34-1600, Invitrogen, Waltham, MA, USA), occludin (1:500; #40-4700, Invitrogen, Waltham, MA, USA) or zonula occludens-1 (ZO-1) (1:750; #40-2200, Invitrogen, Waltham, MA, USA). For detection, horseradish peroxidase conjugated secondary antibodies and enhanced chemiluminescence substrate were applied. Quantification of band intensity was performed using ImageJ 1.52p. For each protein, peak ratios to β-actin were calculated to compare relative expression levels.
7
Western Blot Analysis of Tight Junction Proteins
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Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). A total of 8 μg protein was loaded into a midi Criterion precast protein gel (4%-20%, Bio-Rad), separated by gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad). Blots were blocked with 5% milk powder in PBST (0.1% Tween 20 in PBS) at room temperature for at least 1 h. Afterward, blots were incubated with primary antibodies for beta actin (1:2,000; Cell Signaling Technology), claudin-5 (1:500; 34-1600, Invitrogen), occludin (1:500; 40-4700, Invitrogen), or ZO-1 (1:750; 40-2200, Invitrogen). Horseradish peroxidaseconjugated secondary antibodies and enhanced chemiluminescence substrate were used for detection. Quantification of band intensity was performed using ImageJ 1.52p. Peak ratios to b actin were calculated to compare relative expression levels for each protein.
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