Whole-cell lysate was prepared in ice-cold RIPA buffer with protease inhibitors (Pierce Biotechnology, USA), followed by protein quantification using BCA-assay kit (Pierce Biotechnology, USA). The lysates were resolved in SDS-PAGE and transferred onto a PVDF membrane and blocked in 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20. Appropriate dilutions of primary (1:1000 for LC3B, 1:2000 for Vinculin in 3% BSA-TBST) and HRP conjugated secondary antibodies (1:5000 anti-Rabbit IgG) were used, following which the signal was detected using ClarityMax ECL Kit and documented in Gel Doc™ XR Imaging System (Bio-Rad, USA). ImageJ version 1.49a was used for quantification.
Clarity max ecl kit
Manufactured by Bio-Rad
Sourced in United States
The Clarity Max ECL kit is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. The kit provides a sensitive and stable signal to enable reliable visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc gel imaging system, by Bio-Rad (2 mentions)
Gfp trap agarose beads, by Proteintech (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Gel doc xr imaging system, by Bio-Rad (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Rabbit anti hsp90, by Proteintech (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Mouse monoclonal anti gapdh, by Abcam (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Bio-Rad (1 mentions)
5 protocols using clarity max ecl kit
1
Western Blot Analysis of LC3B and Vinculin
2
GFP-Trap® Protein Interactome Identification
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Cells were washed with PBS, lysed in ice cold IP buffer (1% Triton-X100 in PBS) on ice with periodic vortexing for 15 min. Unbroken cells and cell debris were pelleted at 17,000 ×g for 15 min, and input samples were taken at this point. Cleared cell lysates were split in half and one half was treated with RNase A (100 μg/ml) for 30 min at RT. Lysates were then incubated with GFP-Trap® agarose beads (ChromoTek) for 4 h at 4 °C. Beads were washed four times with ice cold IP buffer, and bound protein complexes were eluted by heating the samples for 10 min at 95 °C in 2xLaemmli loading buffer. Pull-down efficiency was analyzed by western blot. For input, 10% of the final IP sample was loaded on the gel. Proteins were resolved in Mini-Protean® TGX precast gels (Bio-Rad) and transferred to the PVDF membrane (GE Healthcare) by semi-dry blotting. Membranes were blocked in non-fat 4% milk in TBS/T and incubated with primary and HRP-conjugated secondary (GE Healthcare) antibodies. For signal detection, Clarity Max ECL kit and ChemiDoc™ Gel Imaging System (Bio-Rad) were used.
3
Western Blot Analysis of BCKDK Protein
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Total protein extraction was performed using a RIPA Lysis and Extraction Buffer (Thermo Scientific, Waltham, MA, USA) supplemented with cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ (Roche, Basel, Switzerland) phosphatase inhibitor tablets. Total protein extracts (15 µg) were electrophoresed on a 10% SDS-PAGE gel. After transfer and blocking on an Immobilon-P membrane (Millipore, Burlington, MA, USA), the membranes were incubated with the primary antibody, washed, and then incubated with the second peroxidase-conjugated antibody. The reaction was revealed with the Clarity Max ECL kit (Biorad, Hercules, CA, USA). Antibodies used were rabbit anti-Flag (Sigma, Overijse, Belgium; No. F7425) to detect exogenous BCKDK; rabbit anti-Hsp90 (Proteintech, Manchester, UK; No. 13171-1-AP); rabbit anti-BCKDK (Sigma, No. AV52131) to detect both endogenous and exogenous BCKDK; rabbit anti-BCKDH-E1α (E4T3D) (Cell signaling, Danvers, MA, USA; No. 90198); rabbit anti-phospho-BCKDH-E1α (Ser293) (E2V6B) (Cell signaling, No. 40368); and anti-rabbit-IgG HRP-linked (Cell Signaling; No. 7074).
4
NeNaC2 Protein Detection by Western Blot
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For NeNaC2 Western blot, we used the method as previously described in ref. 82 (link). In brief, custom polyclonal antibodies raised against recombinant fragment antigens generated by immunization of rabbits (GenScript, USA). The sequence of the recombinant fragment was ITGCLSLYDLKLIAAVMSCPVAQRHFETEEDKKDEDEDDRAEDPVDENPDDTITVSQMWQDFLHTLTLHGFRFVFERGPTHHHHHH. Equal amounts of protein were run on 4–15% Mini-PROTEAN® TGX™ Precast Protein Gel (Bio-Rad, USA) followed by blotting to a Polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Membrane was incubated overnight with polyclonal antibody against NeNaC2 or monoclonal mouse anti-GAPDH (Abcam, UK) with a dilution of 1:1000 at 4 °C overnight, then washed and incubated for 1 h with peroxidase-conjugated anti-mouse or anti-rabbit antibody (Jackson ImmunoResearch, USA) with a dilution of 1:10,000. Detection was performed with the Clarity™ Max ECL kit (Bio-Rad) according to the manufacturer’s instructions and visualized with a CCD camera of the Odyssey Fc imaging system (Li-COR Biociences, USA).
5
GFP-Trap Protein Complex Purification
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Cells were washed with PBS, lysed in ice cold IP buffer (1% Triton-X100 in PBS) on ice with periodic vortexing for 15 min. Unbroken cells and cell debris were pelleted at 13,000 rpm for 15 min, and input samples were taken at this point. Cleared cell lysates were split in half and one half was treated with RNase A (100 µg/ml) for 30 min at RT. Lysates were then incubated with GFP-Trap® agarose beads (ChromoTek) for 4 h at 4 C. Beads were washed four times with ice cold IP buffer, and bound protein complexes were eluted by heating the samples for 10 min at 95 C in 2xLaemmli loading buffer. Pull-down efficiency was analysed by western blot. For input, 10% of the final IP sample was loaded on the gel. Proteins were resolved in Mini-Protean® TGX precast gels (Bio-Rad) and transferred to the PVDF membrane (GE Healthcare) by semi-dry blotting. Membranes were blocked in non-fat 4% milk in TBS/T and incubated with primary and HRP-conjugated secondary (GE Healthcare) antibodies. For signal detection, Clarity Max ECL kit and ChemiDoc™ Gel Imaging System (Bio-Rad) were used.
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