To discern the conformation of protein aggregation, equal amounts of protein extracts were separated with 4–15% SDS-PAGE under native conditions. Protein extracts were mixed with the sample buffer that does not contain reducing agents and SDS; the samples were not heated and were run in SDS-free Tris Buffer (Bio-Rad). After blocking in 5% nonfat dry milk dissolved in PBST for 1 h, the transferred PVDF membrane (Bio-Rad) was incubated overnight in primary antibodies diluted in 5% nonfat milk or 3% BSA in PBST at 4 °C. After sufficient washes, the membrane was incubated for 1 h at room temperature with HRP-conjugated donkey anti-rabbit or mouse IgG (Cell signaling), treated with chemiluminescence substrate (SuperSignal, Pierce) and exposed on film (Kodak) or imaged using ChemiDo XRS+ (BIO-RAD). Density of bands was measured using ImageJ (http://imagej.nih.gov/ij ).
Chemido xrs
Manufactured by Bio-Rad
Sourced in United States
The ChemiDo XRS+ is a gel documentation and imaging system designed for the detection and analysis of proteins, nucleic acids, and other biomolecules. It features a high-resolution CCD camera, multiple illumination options, and software for image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated donkey anti rabbit or mouse igg, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
P erk, by Abcam (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
2 protocols using chemido xrs
1
Native Protein Aggregation Profiling
2
Spinal Cord Injury Protein Analysis
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Spinal cord was harvested 4 h after SCIRI. Total proteins were extracted with M-PER Mammalian Protein Extraction Reagent (Thermo, USA). The following primary antibodies: GRP78 (1:1000), IRE1α (1:1000, Cell Signaling Technology (CST), USA), ATF6 (1:1000, Abcam), PERK (1:1000, Abcam), CHOP (1:1000, CST), B-cell lymphoma 2 (Bcl-2, 1:3000, Abcam), Bcl-2-associated X, apoptosis regulator (Bax, 1:3000, Abcam), caspase-3 (1:2000, Abcam), and β-actin (1:2500, Abcam). Blots were exposed to the Molecular Imager (ChemiDo XRS+; Bio-Rad, Hercules, USA) and analyzed with ImageJ software (Scion Co., Walkersville, USA).
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