Protein-protein interactions and conformational changes were assessed by luminescence-proximity AlphaScreen technology as described previously52 (link). Protein-protein interaction reactions contained 100 nM recombinant His6-tagged proteins bound to nickel-acceptor beads and 100 nM recombinant biotin-tagged proteins bound to streptavidin donor beads. In the reactions assessing conformational changes, 100 nM recombinant biotin-γ1(residues 24-327)-β2 (residues 76-272)-His6-α1(residues 11-550) was attached to nickel-chelated acceptor beads and streptavidin-coated donor beads. All reactions were performed in buffer containing 50 mM MOPS, pH 7.4, 50 mM NaF, 50 mM CHAPS, and 0.1 mg/ml bovine serum albumin. Where present, bovine liver glycogen (Sigma) had a final concentration of 0.5 mg/ml (∼3 mM glucose units) and cyclodextrin of 2 mM. With the exception of the dose-response curves in Figure 1 , AMP concentrations were fixed at 0.2 mM and ATP concentrations at 1 mM.
Bovine liver glycogen
Manufactured by Merck Group
Bovine liver glycogen is a laboratory product derived from the liver of cattle. It is a form of carbohydrate that serves as an energy storage molecule in animal tissues. The product provides a source of glycogen for various research and analytical applications.
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Lab products found in correlation
Instantblue protein stain, by Abcam (2 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (2 mentions)
Human salivary α amylase, by Merck Group (2 mentions)
Pierce glycoprotein staining kit, by Thermo Fisher Scientific (2 mentions)
Flamingo fluorescent protein stain, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Pullulan, by Merck Group (1 mentions)
Potato amylose, by Merck Group (1 mentions)
Corn amylopectin, by Merck Group (1 mentions)
Potato amylopectin, by Merck Group (1 mentions)
Geldoc go, by Bio-Rad (1 mentions)
4 protocols using bovine liver glycogen
1
Luminescence-based Protein Interaction Assay
2
Non-denaturing PAGE for Protein-Polysaccharide Binding
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Non-denaturing polyacrylamide gels with and without potato amylopectin (Sigma), corn amylopectin (Sigma), potato amylose (Sigma), bovine liver glycogen (Sigma), pullulan (Sigma) or dextran (Sigma) to a final concentration of 0.1% polysaccharide were cast. All polysaccharides were autoclaved, and amylose was solubilized by alkaline solubilization with 1 M NaOH and acid neutralization to pH 7 with HCl 60 . Sas6 protein samples were mixed with 6X loading dye lacking SDS. Gels were run concurrently for 4 h on ice and subsequently stained with Coomassie (0.025% Coomassie blue R350, 10% acetic acid and 45% methanol). Gels were imaged on a Bio-Rad Gel Doc Go imaging system. The distance between each band and the top of the separating gel was measured using ImageJ 61 . The ratio of the distance migrated by each band to the distance the BSA band traveled was determined. Binding was considered positive if the ratio was less than 0.85, as previously described 43 .
3
Ubiquitin Conjugation Assay with Glycogen
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Reactions (20 μl) contained 500 nM His6‐UBE1, 2 μM UBE2L3, 2 μM HOIL‐1, 10 μM Cy5‐ubiquitin (South Bay Bio) and 10 mg/ml bovine liver glycogen (Sigma) in phosphate buffered saline pH 7.4 containing 0.5 mM TCEP and 5 mM Mg2+‐ATP. Reactions were incubated at 37°C for 1 h with gentle mixing. Where indicated, reactions were then incubated in the presence of 50 μg/ml (~0.1 units) human salivary α‐amylase (Sigma) or 1.5 M hydroxylamine for a further 60 min at 37°C. Reactions were stopped by adding NuPAGE LDS sample loading buffer supplemented with 50 mM DTT, and denatured at room temperature for 10 min. Reaction products were separated by gel electrophoresis on 4–12% Bis–Tris gradient gels using MES SDS running buffer (Invitrogen) and stained with Flamingo fluorescent protein stain (Bio‐Rad). Visualisation of the fluorescent signals was performed using the ChemiDoc MP Imaging System (Bio‐Rad) and ImageJ software (Schneider et al, 2012 (link)). In the case of assays using glycogen as substrate, these gels were then stained with periodic acid‐Schiff stain using the Pierce Glycoprotein Staining Kit (Thermo Scientific) and Coomassie stained with InstantBlue Protein Stain (Expedeon).
4
In Vitro Ubiquitination Assay with Glycogen
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Reactions (20 μL) contained 500 nM His 6 -UBE1, 2 μM UBE2L3, 2 μM HOIL-1, 10 μM Cy5-ubiquitin (South Bay Bio) and 10 mg/mL bovine liver glycogen (Sigma) in Phosphate Buffered Saline pH 7.4 containing 0.5 mM TCEP and 5 mM Mg 2+ -ATP. Reactions were incubated at 37°C for 1 h with gentle mixing. Where indicated, reactions were then incubated in the presence of 50 μg/mL (~0.1 units) human salivary α-amylase (Sigma) or 1.5 M hydroxylamine for a further 60 min at 37°C. Reactions were stopped by adding NuPAGE LDS sample loading buffer supplemented with 50 mM DTT, and denatured at room temperature for 10 min. Reaction products were separated by gel electrophoresis on 4-12% Bis-Tris gradient gels using MES SDS running buffer (Invitrogen) and stained with Flamingo fluorescent protein stain (Bio-Rad). Visualisation of the fluorescent signals was performed using the ChemiDoc MP Imaging System (Bio-Rad) and ImageJ software. In the case of assays using glycogen as substrate, these gels were then stained with Periodic acid-Schiff stain using the Pierce Glycoprotein Staining Kit (Thermo Scientific) and Coomassie stained with InstantBlue Protein Stain (Expedeon).
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