The samples were diluted with 10 mM ammonium carbonate buffer pH 7.4 to a concentration of 0.5 mg/mL (protein component mass) and were mixed 4+1 (v/v) with a sample buffer containing 312.5 mM TRIS, 50% (v/v) glycerol, and 0.5 g/l bromophenol blue, pH 6.8. Volumes of 5 µL were loaded onto native 8% polyacrylamide gels with 3.75% stacking gels, which were prepared in a Bio-Rad multicasting chamber using a standard gel casting protocol (consecutive combination of TRIS buffer, acrylamide solution, APS, and TEMED). Electrophoresis was performed for 135 min at 80 V using a Bio-Rad Mini-PROTEAN™ Tetra Cell electrophoresis system with a 25 mM TRIS, 192 mM glycine running buffer. Afterwards, the gels were stained with Coomassie®-staining solution. A calibration curve (2nd order polynomial fit) was generated from the size of each NativeMark™ protein standard (size according to Holzer et al (2017)23 (link)) and the respective distance from the bromophenol blue band. The analyte size was then calculated from the band distance accordingly.
Multicasting chamber
Manufactured by Bio-Rad
The Multicasting chamber is a lab equipment designed for the simultaneous separation and analysis of multiple samples through electrophoresis. It allows for the parallel execution of gel-based experiments, facilitating efficient and high-throughput data generation.
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Lab products found in correlation
2 protocols using multicasting chamber
1
Native Polyacrylamide Gel Electrophoresis
2
Vertical SDS-PAGE Gel Casting and Electrophoresis
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Gels for second-dimension vertical SDS-PAGE were cast using a Bio-Rad multicasting chamber, low-fluorescent glass plates, and 1 mm spacers (Bio-Rad). For a final concentration of the separation gels of 12%, 240 mL acrylamide stock solution (40%, T : C = 29 : 1, Bio-Rad) was mixed with 200 mL 1.5 M Tris-HCl pH 8.8, 40 mL glycerol, and 320 mL H2OUHQ. TEMED (80 μL) and ammonium persulfate (1 mL, APS, 10% in H2OUHQ) were added after degassing of the mixture and right before filling of the casting chamber. The gels were left to polymerize overnight, overlaid with water-saturated n-butanol. Vertical second-dimension SDS-PAGE was carried out on a Bio-Rad Dodeca system with the current set to 60 mA for 100 Vh and then to 200 mA for 1200 Vh.
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