SDS-PAGE was carried out using precast 4-20% gradient polyacrylamide gels (Expedeon) and stained with InstantBlue (Expedeon). The PageRuler Prestained NIR Protein Ladder (Thermo Scientific) was included. Proteins were transferred to PVDF membranes (Millipore) in run blot buffer (Expedeon) and 20% methanol (VWR), washed in TBS-T buffer and blocked in TBS-T with 5% skim milk (Sigma). Blots were incubated overnight at 4°C with agitation in blocking buffer and anti-HisG-HRP (1:5000, Invitrogen), or anti-V5-HRP (1:5000, Invitrogen). Chemiluminescent detection of blots was conducted with Luminata Forte (Millipore) in a G-box illuminator with Epi Red and FRLP filters (Syngene) for visualizing ladder and samples, respectively.
Gradient polyacrylamide gels
Manufactured by Abcam
4-20% gradient polyacrylamide gels are laboratory equipment used for protein separation and analysis. They are composed of a gradient of polyacrylamide concentrations ranging from 4% to 20%, which allows for the separation of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Methanol, by Avantor (2 mentions)
Skim milk, by Merck Group (2 mentions)
Luminata forte, by Merck Group (2 mentions)
Instantblue, by Abcam (2 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (2 mentions)
Pageruler prestained nir protein ladder, by Thermo Fisher Scientific (2 mentions)
Anti hisg hrp, by Thermo Fisher Scientific (2 mentions)
Epi red, by Syngene (2 mentions)
Run blot buffer, by Abcam (2 mentions)
2 protocols using gradient polyacrylamide gels
1
Comprehensive Protein Visualization Workflow
2
Protein Detection and Quantification
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SDS-PAGE was carried out using precast 4-20% gradient polyacrylamide gels (Expedeon) and stained with InstantBlue (Expedeon). The PageRuler Prestained NIR Protein Ladder (Thermo Scientific) was included. Proteins were transferred to PVDF membranes (Millipore) in run blot buffer (Expedeon) and 20% methanol (VWR), washed in TBS-T buffer and blocked in TBS-T with 5% skim milk (Sigma). Blots were incubated overnight at 4C with agitation in blocking buffer and anti-HisG-HRP (1:5000, Invitrogen), or anti-V5-HRP (1:5000, Invitrogen). Chemiluminescent detection of blots was conducted with Luminata Forte (Millipore) in a Gbox illuminator with Epi Red and FRLP filters (Syngene) for visualizing ladder and samples, respectively.
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