To separate PB‐α2AP and NPB‐α2AP, 5 µg (for SDS‐PAGE) and 300 ng (for Western blot analyses) affinity‐purified α2AP in Tris‐buffered saline were boiled for 5 to 10 minutes at 95°C in the presence of XT Sample Buffer and XT reducing agent and resolved on a precast XT Criterion 10% polyacrylamide gel. After electrophoresis, the proteins were stained by Coomassie or transferred to a nitrocellulose membrane using a PowerPac™ HC power supply (BioRad, Richmond, CA) with transfer buffer (25 mmol/L Tris, 192 mmol/L glycine [pH 8.3], and 20% methanol) at 100V for 1 hour. After protein transfer, nonspecific sites on the nitrocellulose membrane were blocked with 5% milk in PBS, pH 7.4, followed by three wash steps with PBS/0.1% Tween‐20. Postblocking, the blot was incubated with a mixture of TC 3AP (1 mg/mL, 1:5000) and anti‐Asn‐α2AP (1 mg/mL, 1:5000) in PBS/0.1% Tween‐20 overnight at 4°C under constant motion. After three wash steps, IRDye® 680CW donkey anti‐mouse secondary antibody and IRDye® 800CW donkey anti‐rabbit secondary antibody were used for detection of TC 3AP and the anti‐Asn‐α2AP antibody, respectively. After incubation for 1 hour at RT, three wash steps were performed and the blot was scanned in the 680‐ or 800‐nm channel of an Odyssey® Imaging System (Lincoln, NE).
Powerpac hc power supply
Manufactured by Bio-Rad
Sourced in United States
The PowerPac™ HC power supply is a laboratory instrument designed to provide a consistent and reliable power source for various electrophoresis applications. It features adjustable voltage, current, and power settings to accommodate a wide range of experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean 3 cell, by Bio-Rad (1 mentions)
Quantipro, by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Protein ladder, by Benchmark Scientific (1 mentions)
Xt sample buffer, by Bio-Rad (1 mentions)
Xt reducing agent, by Bio-Rad (1 mentions)
4 protocols using powerpac hc power supply
1
Purification and Western Blot Analysis of α2AP
2
Infliximab Stability after Microencapsulation
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The stability of infliximab’s primary structure after microencapsulation was assessed by SDS-PAGE electrophoresis under reducing and non-reducing conditions. Before analysis, the infliximab content of samples and standards was determined using a BCA protein assay kit (QuantiPro; Sigma-Aldrich), and concentrations were equalized. Afterwards, 10 µl of infliximab samples and standards (200 µg/ml) was mixed with 10 µl of Laemmli sample buffer containing or not 5% β-mercaptoethanol for reducing or non-reducing environment, respectively, and then heat denatured at 95 °C for 35 min. Subsequently, SDS-PAGE analysis was performed on a 13% T (Total concentration of acrylamide plus bis-acrylamide) resolving gel (1.5 M Tris–HCl, pH 8.8) with a 5% T (0.5 M Tris–HCl; pH 6.8) stacking gel at a constant voltage of 200 V for 35 min using a Mini-PROTEAN® 3 cell equipped with a PowerPac™ HC Power Supply (Bio-Rad Laboratories, Hercules, CA, USA). For molecular weight determination, Precision Plus Dual Color Protein Standards were used (Bio-Rad). All analyses were carried out in triplicate. Finally, gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad), and images were then recorded with a GD1000 Axygen® Gel Documentation System (Corning Inc, Corning, NY, USA). Further analysis was performed using GelAnalyzer 19.1 software (available at http://www.gelanalyzer.com ).
3
SDS-PAGE Protein Separation and Visualization
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SDS-PAGE was performed on a vertical system Mini-PROTEAN® Tetra Cell with a PowerPac™ HC Power Supply (Bio-Rad, Hercules, USA) using in-house 10% SDSpolyacrylamide gels (30% (v/v) Acrylamide/bis solution, 0.375 M Tris-HCl pH 8.8, 10% (v/v) SDS, 10% (v/v) APS and 0.004 % (v/v) TEMED). Fifteen µL of each IAC purified serum sample or mouse apo-transferrin standard (~10 µg of protein) were reduced and denatured with 5 µL of reducing-Laemmli sample buffer (0.25 M Tris-HCl pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 10% (v/v) β-ME and 1% (v/v) bromophenol blue), incubating the mixture in a thermoshaker at 100ºC for 5 min. Then, samples were loaded into the gel to perform the protein separation. Ten µL of Protein Ladder (BenchMark™ Protein Ladder) were also loaded in one lane in order to assign the molecular weight to the bands. Gel electrophoresis was performed at 120 V for 2 h at room temperature using a running buffer consisted of 25 mM Tris-base, 250 mM glycine and 0.1% SDS. After SDS-PAGE, gel was fixed in 40% (v/v) ethanol and 10% (v/v) HAc for 30 min and then rinsed in Milli-Q water (3 x 5 min). Gel was incubated with Coomassie blue staining solution at room temperature for 1 h with agitation, and then rinsed in Milli-Q water until a proper degree of staining was achieved.
4
Western Blot Analysis of α2AP Variants
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To investigate the reactivities of the mAbs by Western blot, recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP in TBS were boiled for five to ten minutes at 95°C in the presence of XT Sample Buffer and XT reducing agent (BioRad, Richmond, CA) and resolved on a 10% SDS-polyacrylamide gel (300 ng/lane). After electrophoresis the proteins were transferred to a nitrocellulose membrane using a PowerPac™ HC power supply (BioRad) with transfer buffer (25 mM Tris, 192 mM glycine (pH 8.3) and 20% methanol) at 100V constant voltage for one hour. After protein transfer, non-specific sites on the nitrocellulose membrane were blocked with 5% milk in PBS, pH 7.4 followed by three washings in PBS with 0.1% Tween-20. Post-blocking, the blots were incubated with selected purified mAbs (800 ng/ml) in PBS with 0.1% Tween-20 for 18 hours under constant motion at 4°C. After three washes with PBS with 0.1% Tween-20, IRDye® 800CW donkey anti-mouse secondary antibodies (1 mg/ml) (Lincoln Nebraska,USA) were added at an optimized dilution of 1:10000 and incubated for one hour at RT. Following three washes with PBS, the blots were scanned in the 800 nm channel of an Odyssey® Imaging System (Lincoln Nebraska,USA).
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