Infrared fluorescent western blotting was conducted to confirm the expression of both GAP and COP in plants. Purified recombinant proteins mixed with 4 μL of 5× loading buffer were electrophoresed using 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA) using the Mini-Protean IITM system (Bio-Rad, Hercules, CA, USA). Membranes were incubated in TBS buffer for 4 h at RT, followed by incubation with the goat anti-human IRDye 800 CW and goat anti-mouse 680 LT (1:15,000; LI-COR, Lincoln, NE, USA) in the blocking buffer at RT for 1.5 h to detect GAP and COP, respectively. After washing four times for 5 min. each in 1 × TBS at RT, the membranes were scanned using the OdysseyTM CLx infrared imaging system (LI-COR).
Mini protean iitm system
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protean IITM system is a vertical electrophoresis system designed for the separation of proteins and nucleic acids. The system features a compact and modular design, allowing for flexibility in the number of samples that can be run simultaneously. It is compatible with a variety of gel chemistries and can be used for a range of applications, including SDS-PAGE, native PAGE, and isoelectric focusing.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse 680lt, by LI COR (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Goat anti human irdye 800 cw, by LI COR (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
4 protocols using mini protean iitm system
1
Infrared Fluorescent Western Blotting for GA^P and CO^P
2
Western Blotting Protocol for Whole Cell Extracts
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For whole cell extracts, cells pellets were lysed in Laemmli buffer 2X (125 mM Tris–HCl pH 6.8; 20% glycerol; 4% SDS; 0.2 M DTT and 1% bromophenol blue). Samples were loaded at 100 000 cells/lane and separated by electrophoresis under denaturing conditions using the Mini-Protean IITM system (BioRad) for 50 min at 180 V. A commercial protein mixture (Dual Color, BioRad) was used as a molecular weight standard. After finishing the electrophoresis, the proteins present in the polyacrylamide gels were transferred to nitrocellulose membranes at 90 V for 45 min. Next, membranes were blocked with 4% (w/v) skimmed milk in TTBS (15 mM Tris–HCl pH 7.5, 200 mM NaCl and 0.1% Tween 20) for 1 h at room temperature. Primary antibodies are listed in ‘antibodies’. Membranes were washed 3 times for 5 min in TTBS, incubated with HRP-conjugated secondary antibody in 4% (w/v) skimmed milk in TTBS and visualized using SuperSignal West Pico PLUS (Thermo Fisher Scientific).
3
Western Blot Detection and Analysis
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Proteins were separated on SDS-PAGE under denaturing conditions and transferred to nitrocellulose membrane using a MINI PROTEAN II TM system (Bio-Rad). Membranes were blocked with 5% milk powder/TBS-T and incubated with the primary antibodies overnight at 4 °C, followed by washing in TBS-T and then HRP-linked secondary antibodies at room temperature for 1 h. Membranes were scanned using a ChemiDoc TM MP Imaging System (Bio-Rad) and images were processed and further analysed using Image Lab Software (Bio-Rad).
4
Western Blot Protein Detection Protocol
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For whole cell extracts, cells pellets were lysed in Laemmli buffer 2X (125 mM Tris-HCl pH 6.8; 20 % glycerol; 4 % SDS; 0.2 M DTT and 1 % bromophenol blue). Samples were loaded at 100000 cells/lane and separated by electrophoresis under denaturing conditions using the Mini-Protean IITM system (BioRad) for 50 minutes at 180 V. A commercial protein mixture (Dual Color, BioRad) was used as a molecular weight standard. After finishing the electrophoresis, the proteins present in the polyacrylamide gels were transferred to nitrocellulose membranes at 90 V for 45 minutes. Next, membranes were blocked with 4% (w/v) skimmed milk in TTBS (15 mM Tris-HCl pH 7.5, 200 mM NaCl and 0.1 % Tween 20) for 1 hour at room temperature. Primary antibodies are listed in "antibodies". Membranes were washed 3 times for 5 minutes in TTBS, incubated with HRP-conjugated secondary antibody in 4% (w/v) skimmed milk in TTBS and visualized using SuperSignal West Pico PLUS (Thermo Fisher Scientific).
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